COMPOSITIONS AND METHODS FOR INHIBITING AN INFLAMMATORY RESPONSE AND TREATING INFLAMMATORY DISEASES
In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS), thus also inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, thus also treating, ameliorating or preventing neutrophil (PMN)-meditated inflammatory-related pathologies such as acute respiratory distress syndrome (ARDS), including ARDS caused by a viral infection such as COVID-19.
This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. § 119(●) of U.S. Provisional Application No. 63/194,502, May 28, 2021. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCHThis invention was made with government support under GM111716 and GM057846 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.
TECHNICAL FIELDThis invention generally relates to biology and inflammatory diseases. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS), thus also inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, thus also treating, ameliorating or preventing neutrophil (PMN)-meditated inflammatory-related pathologies such as acute respiratory distress syndrome (ARDS), including ARDS caused by a viral infection such as an influenza or a coronavirus infection, for example, caused by infection with COVID-19.
BACKGROUNDNeutrophils, or polymorphonuclear leukocytes (PMNs), release reactive oxygen species (ROS) to combat infection, but this inflammatory response can also produce pathology such as acute respiratory distress syndrome (ARDS), a disorder that leads to accumulation of albumin-rich fluid in the lung air spaces and is fatal in 40% of patients. Pharmacologic therapies focused on downstream cytokine actions have failed to improve morbidity or mortality.
Human voltage-gated proton channels (hHv1) are expressed in many human tissues including innate and adaptive immune cells, cancer cells and sperm. hHv1 channels are comprised of two identical subunits, each with 273 residues and four transmembrane spans (S1-S4) that resemble the voltage sensor domains (VSDs) in conventional voltage-gated ion channels. In hHv1, there are two H+-selective conduction pathways, one in each VSD. The activation of hHv1 depends on both the transmembrane potential and the pH gradient across the membrane. Upon membrane depolarization, hHv1 S4 segments move outward leading to conformational changes that open the H+ conduction pathways.
hHv1 has been implicated in many aspects of health and disease. In the absence of known high-affinity and specific inhibitors of the channel, we designed a selective inhibitor of hHv1, the C6 peptide, and used it to demonstrate that H+ efflux via the channel is required in human sperm to induce intracellular alkalization and Ca2+ influx to initiate capacitation. Furthermore, we showed that it also operates in human neutrophils to maintain cytoplasmic pH during the respiratory burst allowing sustained reactive oxygen species (ROS) production. We identified C6 using a high-throughput, phage-display strategy whereby approximately 1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) toxin scaffold and sorted on purified hHv1 protein. Phagemids expressing C6 were selected by their capacity to bind to hHv1 protein. C6 has 41 residues including six cysteines that form three intramolecular disulfide bonds.
Before the development of C6, known hHv1 inhibitors were pharmacologically promiscuous or of low affinity. Synthesized C6 inhibits hHv1 by binding with positive cooperatively to the external channel residues linking the S3 and S4 transmembrane spans (S3-S4 loop), one peptide on each loop, and holds the voltage sensor in a conformation that favors channel closure. As a result, the channel passes fewer H+ ions because more positive voltages are required to open the pores.
SUMMARYIn alternative embodiments, provided are synthetic peptides (also called Hv1 modulating agents) for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide has or consists of:
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- (a) an amino acid sequence QXHQFEXXXX (SEQ ID NO:1);
- (b) an amino acid sequence QAHQFEAXXX (SEQ ID NO:2);
- (c) an amino acid sequence QAHQFEALLL (SEQ ID NO:3), or
- (d) a homodimer or a heterodimer comprising a peptide of (a), (b), or (c), wherein optionally the heterodimer comprises (a) and (b), (b) and (c), or (a) and (c), or the homodimer comprise two of (a), two of (b) or two of (c), or a trimer comprising three of (a), (b) or (c), or any combination thereof,
- wherein X is any hydrophobic amino acid, and optionally the hydrophobic amino acid is glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) or phenylalanine (F).
In alternative embodiments, provided are recombinant nucleic acids encoding a synthetic peptide as provided herein.
In alternative embodiments, provided are expression vehicles comprising or having contained therein a recombinant nucleic acid as provided herein, wherein optionally the expression vehicle comprises or is an expression cassette, a plasmid, an expression vector or a recombinant virus.
In alternative embodiments, provided are cells comprising or having contained therein a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, or an expression vehicle as provided herein.
In alternative embodiments, provided are pharmaceutical compositions comprising: a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, or an expression vehicle as provided herein. In alternative embodiments, the pharmaceutical compositions are formulated as a liposome, a dendrimer or a nanoparticle; or formulated as an aerosol, a lyophilate or a powder.
In alternative embodiments, provided are methods for:
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- inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm,
- inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS),
- inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, or
- treating, ameliorating or preventing a neutrophil (PMN)-meditated inflammatory-related pathology,
- comprising:
- administering to an individual in need thereof:
- (a) a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, an expression vehicle as provided herein, a cell as provided herein or a pharmaceutical composition as provided herein, or
- (b) a synthetic peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide has or consists of a structure:
- (i) of about 10 to about 100 amino acids in length;
- (ii) having an amino acid sequence that comprises one or more toxin sequence elements, each of which has a length within a range of about 5 to about 18 amino acids in length;
- (iii) a peptide component has 0, 1, 2, 3, 4, 5, or 6 conserved cysteine residues of an ICK structural motif,
- (iv) has 0, 1, 2, or 3 disulfide bridges; and
- (v) has 0, 1, 2, or 3 beta strands,
- wherein optionally the synthetic peptide has a sequence comprising or consisting of:
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- a homodimer comprising two peptides (SEQ ID NO:4) (also called “C6”) or two peptides (SEQ ID NO:5) (also called “C5”), or a heteroduplex of (SEQ ID NO:4) and (SEQ ID NO:5),
- or a sequence as described in U.S. patent application publication no: US 2019 0330285 A1.
In alternative embodiments of the methods:
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- the neutrophil (PMN)-meditated inflammatory-related pathology is acute respiratory distress syndrome (ARDS),
- and optionally ARDS caused by a viral, bacterial or fungal infection; a sepsis; pancreatitis; trauma or injury; pneumonia; or, aspiration into the lungs,
- and optionally the viral infection is an influenza or a coronavirus infection, and optionally the coronavirus infection is a COVID-19 infection; and/or
- the synthetic peptide is formulated and/or administered as a liposome, a dendrimer or a nanoparticle; or formulated or administered as an aerosol, a lyophilate or a powder.
In alternative embodiments, provided are kits comprising a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, an expression vehicle as provided herein, a pharmaceutical composition as provided herein, or a cell as provided herein, or a synthetic peptide as used in a method of any of the preceding claims, and optionally including instructions for practicing a method of any of the preceding claims.
In alternative embodiments, provided are uses of:
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- (a) a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, an expression vehicle as provided herein, a pharmaceutical composition as provided herein, or a cell as provided herein, or
- (b) a synthetic peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide has or consists of a structure:
- (i) of about 10 to about 100 amino acids in length;
- (ii) having an amino acid sequence that comprises one or more toxin sequence elements, each of which has a length within a range of about 5 to about 18 amino acids in length;
- (iii) a peptide component has 0, 1, 2, 3, 4, 5, or 6 conserved cysteine residues of an ICK structural motif,
- (iv) has 0, 1, 2, or 3 disulfide bridges; and
- (v) has 0, 1, 2, or 3 beta strands,
- wherein the synthetic peptide has a sequence comprising or consisting of:
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- or a sequence as described in U.S. patent application publication no: US 2019 0330285 A1,
- in the preparation of a pharmaceutical composition for:
- inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm,
- inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS),
- inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, or
- treating, ameliorating or preventing a neutrophil (PMN)-meditated inflammatory-related pathology.
In alternative embodiments, provided are synthetic peptides for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide has or consists of:
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- (a) an amino acid sequence QXHQFEXXXX (SEQ ID NO: 1);
- (b) an amino acid sequence QAHQFEAXXX (SEQ ID NO:2);
- (c) an amino acid sequence QAHQFEALLL (SEQ ID NO:3), or
- (d) a homodimer comprising a peptide of (a), (b), or (c),
- wherein X is any hydrophobic amino acid, and optionally the hydrophobic amino acid is glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) or phenylalanine (F).
In alternative embodiments of methods as provided herein:
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- the neutrophil (PMN)-meditated inflammatory-related pathology is acute respiratory distress syndrome (ARDS), and optionally ARDS caused by a viral, bacterial or fungal infection; a sepsis; pancreatitis; trauma or injury; pneumonia; or, aspiration into the lungs, and optionally the viral infection is an influenza or a coronavirus infection, and optionally the coronavirus infection is a COVID-19 infection; and/or
- the synthetic peptide is formulated and/or administered as a liposome, a dendrimer or a nanoparticle; or formulated or administered as an aerosol, a lyophilate or a powder.
In alternative embodiments, provided are synthetic peptides for use in:
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- inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm,
- inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS),
- inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, or
- treating, ameliorating or preventing a neutrophil (PMN)-meditated inflammatory-related pathology, or
- for use in the preparation of a pharmaceutical composition, wherein the synthetic peptide comprises:
- (a) a synthetic peptide as provided herein, a recombinant nucleic acid as provided herein, an expression vehicle as provided herein, a pharmaceutical composition as provided herein, or a cell as provided herein, or
- (b) a synthetic peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide has or consists of a structure:
- (i) of about 10 to about 100 amino acids in length;
- (ii) having an amino acid sequence that comprises one or more toxin sequence elements, each of which has a length within a range of about 5 to about 18 amino acids in length;
- (iii) a peptide component has 0, 1, 2, 3, 4, 5, or 6 conserved cysteine residues of an ICK structural motif;
- (iv) has 0, 1, 2, or 3 disulfide bridges; and
- (v) has 0, 1, 2, or 3 beta strands,
- wherein the synthetic peptide has a sequence comprising or consisting of:
-
- or a sequence as described in U.S. patent application publication no: US 2019 0330285 A1.
The details of one or more exemplary embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference in their entireties for all purposes.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
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SEQ ID NO:6 is hHv1: VVSFILDIVLLFQEHQFEALGLLILLRLWRVARII
SEQ ID NO:12 is hKv2.1: LPYYVTIFLTESNKSVLQFQNVRRVVQIFRIMRIRILKLA
SEQ ID NO:13 is hS3-S4Kv2.1: LPYYILDIVLLFQEHQFEALGLLILLRLWRILKL, and
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Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are compositions comprising a synthetic peptide as provided herein, and products of manufacture and kits, and methods, for inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS), thus also inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, thus also treating, ameliorating or preventing neutrophil (PMN)-meditated inflammatory-related pathologies such as acute respiratory distress syndrome (ARDS), including ARDS caused by a viral infection such as COVID-19.
In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm.
In alternative embodiments, provided are compositions and methods for targeting human hHv1 in PMN because (i) the channel in these cells initiates and sustains ARDS, (ii) C6 (SEQ ID NO:4), a unique blocker of hHv1 suppresses ROS production by human PMN5 and (iii) C6 suppresses pulmonary compromise in an acute lung injury mouse model for ARDS.
A high-affinity and specific direct blocker of Hv1 (C6 peptide) has been used to show, first, that human sperm require hHv1-mediated H+ efflux to initiate capacitation, allowing the acrosomal reaction, and oocyte fertilization; and, second, that hHv1 in human PMN is required to produce and sustain release of inflammatory agents, including ROS and proteases, during the innate immune inflammatory response. Data as provided herein demonstrate a second target in the pathway: albumin (Alb) is required to activate hHv1 in human PMN and we describe a peptide (L*) that blocks Alb-activation and ROS production. Supporting our driving hypothesis, we show here that both C6 and L* inhibit hHv1 in human PMN, decreasing ROS production, and that C6 protects in an acute lung-injury mouse model, restoring lung compliance, and decreasing ROS, cytokines, protein, and cells in bronchoalveolar lavage fluid. We also present new, novel methods to speed structure-function studies and inhibitor design (T-peptides) and screen for small molecule inhibitors. We benefit from use of cutting-edge biophysical, spectroscopic, structural, and in vivo methods, and two expert collaborators.
Alb activation of hHv1 seeks the structural and mechanistic basis for the action of Alb and a more potent natural metabolite. Alb regulation of the PMN inflammatory response delineates the role of hHv1 in PMN using C6 (SEQ ID NO:4) and the peptide QAHQFEALLL (SEQ ID NO:3), also called “L*”. Inhibiting PMN inflammatory pathology seeks the basis for action of C6 and L*; tests second generation peptide inhibitors and small molecule mimetics; and studies select agents on human PMN and an ARDS mouse model.
Significance: Embodiments as provided herein address an unmet medical need, recently made more apparent by the advent of COVID-19-related ARDS, and has broader influence because Hv1 and Alb in PMN and other cells are complicit in additional acute and chronic inflammatory disorders. Unique hHv1 inhibitors and innovative methods are applied herein to understand and suppress the pathophysiology of ARDS. Embodiments as provided herein address a specific unmet medical need, the absence of an effective medical therapy for Acute Respiratory Distress Syndrome (ARDS), a disorder that is fatal in 40% of patients (and is seen, for example, in patients with SARS-CoV-2 infection). Embodiments as provided herein are broadly impactful because the white blood cells called neutrophils (PMN) that damage the lungs in ARDS also cause other acute and chronic inflammatory diseases. We have showed that the human voltage-gated proton channel (hHv1) in PMN is essential to produce the inflammatory response, and provided herein is a treatment for ARDS by a novel strategy: targeting hHv1 in PMN, both directly and by blocking a natural activator of hHv1 we describe here to be required for normal hHv1 operation, thereby offering two ways to confront the disease at the earliest stage before it becomes a more complex disorder.
We designed C6 peptide to address the absence of specific inhibitors of human voltage-gated proton channels (hHv1). Two C6 bind to the two hHv1 voltage sensors at the resting state, inhibiting activation on depolarization. As discussed in Example 4, we identified the C6-hHv1 binding interface using tethered-toxin variants and channel mutants, unveiling an important role for negatively-charged lipids and present a model of the C6-hHv1 complex. We created a peptide with two C6 epitopes (C62) that binds to both channel subunits simultaneously, yielding picomolar affinity and significantly improved inhibition at high potentials. C6 and C62 are novel tools to regulate hHv1, a channel involved in innate immune system inflammatory pathophysiology, sperm capacitation, cancer cell proliferation, and tissue damage in ischemic stroke.
Pharmaceutical CompositionsIn alternative embodiments, provided are pharmaceutical compositions comprising a synthetic peptide as provided herein, a recombinant nucleic acid, or an expression vehicle as provided herein, wherein optionally the pharmaceutical composition is formulated as a liposome, a dendrimer or a nanoparticle; or formulated as an aerosol, a lyophilate or a powder. In alternative embodiments, pharmaceutical compositions are provided herein are administered for: inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm; inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS); inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response; and/or treating, ameliorating or preventing a neutrophil (PMN)-meditated inflammatory-related pathology.
In alternative embodiments, pharmaceutical composition as used herein comprises a composition that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to show a statistically significant probability of achieving a desired therapeutic effect when administered to a subject or population in need thereof. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, for example, those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces. In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is sterile and substantially pyrogen-free.
In some embodiments, a composition as provided herein comprises a pharmaceutically acceptable composition that includes and/or delivers a synthetic peptide as provided herein, for example, an Hv1 modulating agent as described herein. For example, in some embodiments, a provided composition includes an Hv1 modulating agent polypeptide component. Alternatively or additionally, in some embodiments, a provided composition includes a nucleic acid that encodes an Hv1 modulating agent polypeptide component, a cell that expresses (or is adapted to express) an Hv1 modulating agent polypeptide component, etc. In some embodiments Hv1 modulating agents having any of the modifications of the present disclosure are included in pharmaceutical compositions.
In some embodiments, general considerations in the formulation and manufacture of pharmaceutical agents are found, for example, in Remington's Pharmaceutical Sciences, 19.sup.th ed., Mack Publishing Co., Easton, Pa., 1995.
In alternative embodiments, pharmaceutical compositions as provided herein are in a variety of forms: these include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (for example, injectable and infusible solutions), dispersions or suspensions, microemulsions, liposomes and suppositories. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. The preferred form of pharmaceutical composition depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for administration of antibodies to humans.
In alternative embodiments, a pharmaceutical composition as provided herein can include a pharmaceutically acceptable carrier. For example, pharmaceutical compositions can include a therapeutic agent in addition to one or more inactive agents such as a sterile, biocompatible carrier.
In alternative embodiments, exemplary carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. For example, carriers may include sterile water, saline, buffered saline, or dextrose solution. In alternative embodiments, the composition contains any of a variety of additives, such as stabilizers, buffers, excipients (for example, sugars, amino acids, etc.), or preservatives. Preferably, the carrier is suitable for oral, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion).
In alternative embodiments, pharmaceutical compositions as provided herein include a pharmaceutically acceptable salt, for example, a salt that retains the desired biological activity of the Hv1 modulating agent and does not impart any undesired toxicological effects (see for example, Berge, S. M., et al., J. Pharm. Sci. 66:1-19, 1977). Depending on the route of administration, the Hv1 modulating agent may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. In certain embodiments, a pharmaceutical composition can include a therapeutic agent that is encapsulated, trapped, or bound within a lipid vesicle, a bioavailable and/or biocompatible and/or biodegradable matrix, or other microparticles.
In certain embodiments, synthetic peptide as provided herein, for example, an Hv1 modulating agent, is prepared with a carrier that protects against rapid release, such as a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid. Many methods for the preparation of such formulations are patented or generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical formulation is a well-established art, and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20.sup.th ed., Lippincott, Williams & Wilkins, 2000; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7.sup.th Ed., Lippincott Williams & Wilkins Publishers, 1999; and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed., 2000.
In alternative embodiments, pharmaceutical compositions as provided herein comprise a synthetic peptide as provided herein, for example, an Hv1 modulating agent, that is not aggregated. For example, in some embodiments, less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, of Hv1 modulating agent is present in an aggregate. In alternative embodiments, pharmaceutical compositions as provided herein comprise an Hv1 modulating agent that is not denatured. For example, less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, of Hv1 modulating agents administered is denatured. In some embodiments, a provided pharmaceutical composition will include an Hv1 modulating agent that is not inactive. For example, less than 1%, 2%, 5%, 10%, 20%, or 30%, by dry weight or number, of Hv1 modulating agents administered is inactive.
In alternative embodiments, pharmaceutical compositions as provided herein are formulated to reduce immunogenicity of provided synthetic peptides as provided herein, for example, Hv1 modulating agents. For example, in some embodiments, a synthetic peptide as provided herein, or Hv1 modulating agent, is associated with (for example, bound to) an agent, such as polyethylene glycol and/or carboxymethyl cellulose, that masks its immunogenicity. In some embodiments, a provided binding agent has additional glycosylating that reduces immunogenicity.
Methods of AdministrationIn alternative embodiments, pharmaceutical compositions as provided herein are administered in any dose appropriate to achieve a desired outcome. In some embodiments, the desired outcome is reduction in intensity, severity, and/or frequency, and/or delay of onset of one or more symptoms of an Hv1 associated disease or condition.
In alternative embodiments, pharmaceutical compositions as provided herein are administered in a therapeutically effective amount of a synthetic peptide as provided herein, for example, a synthetic peptide as provided herein, or an Hv1 modulating agent composition, for example, an amount which is effective, upon single or multiple dose administration to a subject, in treating a subject, for example, curing, alleviating, relieving or improving at least one symptom of a disease or condition in a subject to a degree beyond that expected in the absence of such treatment. A therapeutically effective amount of the composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition is outweighed by the therapeutically beneficial effects. A therapeutically effective dosage can modulate a measurable parameter, favorably, relative to untreated subjects. The ability of a synthetic peptide as provided herein, or an Hv1 modulating agent, to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in a human disorder.
In some embodiments, pharmaceutical compositions are administered in multiple doses. In some embodiments, pharmaceutical compositions are administered in multiple doses/day. In some embodiments, pharmaceutical compositions are administered according to a continuous dosing regimen, such that the subject does not undergo periods of less than therapeutic dosing interposed between periods of therapeutic dosing. In some embodiments, pharmaceutical compositions are administered according to an intermittent dosing regimen, such that the subject undergoes at least one period of less than therapeutic dosing interposed between two periods of therapeutic dosing.
Dosage regimens can be adjusted to provide the optimum desired response (for example, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of ligand calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a synthetic peptide as provided herein, for example, an Hv1 modulating agent described herein, is between about 0.1-20 mg/Kg, or about 1-10 mg/Kg. In some embodiments, an agent can be administered by parenteral (for example, intravenous or subcutaneous) infusion at a rate of less than 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 50 mg/m.sup.2 or about 5 to 20 mg/m2. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions (for example, the supervising physician), and that dosage ranges set forth herein are only exemplary.
In alternative embodiments, pharmaceutical compositions as provided herein are administered by a variety of routes, including oral, intravenous, intramuscular, intra-arterial, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, or drops), mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. For example, for therapeutic applications, a synthetic peptide as provided herein, for example, an Hv1 modulating agent composition, can be administered by intravenous infusion at a rate of less than 30, 20, 10, 5, or 1 mg/min to reach a dose of about 1 to 100 mg/m.sup.2 or 7 to 25 mg/m.sup.2. Alternatively, the dose could be 100.mu.g/Kg, 500.mu.g/Kg, 1 mg/Kg, 5 mg/Kg, 10 mg/Kg, or 50 mg/Kg. The route and/or mode of administration will vary depending upon the desired results. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (for example, its stability in the environment of the gastrointestinal tract), the condition of the patient (for example, whether the patient is able to tolerate oral administration), etc.
In alternative embodiments, pharmaceutical compositions as provided herein are administered by: parenteral (for example, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion) means. In one embodiment, the Hv1 modulating agent composition is administered by intravenous infusion or injection. In another embodiment, the Hv1 modulating agent composition is administered by intramuscular or subcutaneous injection. In another embodiment, the Hv1 modulating agent composition is administered orally. In some embodiments, the Hv1 modulating agent composition is administered topically. In some embodiments, the Hv1 modulating agent composition is administered transdermally. Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage.
In alternative embodiments, pharmaceutical compositions as provided herein comprise a synthetic peptide as provided herein, for example, Hv1 modulating agents, that are administered either alone or in combination with one or more other agents. In some embodiments, Hv1 modulating agents or pharmaceutical compositions of the present disclosure may be administered with one or more other Hv1 modulating agents. In some embodiments, Hv1 modulating agents or pharmaceutical compositions of the present disclosure may be administered with one or more other pharmaceutical agent including, but not limited to, small molecules, vaccines and/or antibodies. In some embodiments, Hv1 modulating agents or pharmaceutical compositions may be administered in combination with an adjuvant. Combinations of agents may be administered at the same time or formulated for delivery together. Alternatively, each agent may be administered at a dose and on a time schedule determined for that agent. Additionally, the invention encompasses the delivery of the pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body. Although the pharmaceutical compositions of the present invention can be used for treatment of any subject (for example, any animal, or a human) in need thereof, they are most preferably used in the treatment of humans.
In alternative embodiments, pharmaceutical compositions as provided herein are used to impact various diseases, disorders, and conditions. In alternative embodiments, pharmaceutical compositions as provided herein are used in the treatment of Hv1 associated diseases or conditions. Synthetic peptides as provided herein, for example, Hv1 modulating agents and/or Hv1 modulating agent compositions described herein, can be administered, alone or in combination with, another agent to a subject, for example, a patient, for example, a patient who has a disorder (for example, an Hv1-associated disease or condition, for example. immune deficiency), a symptom of a disorder or a predisposition toward a disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder. The treatment may also delay onset, for example, prevent onset, or prevent deterioration of a condition.
In some embodiments, synthetic peptides as provided herein, or Hv1 modulating agents, formulated in pharmaceutical compositions, are administered to a subject suffering from or susceptible to an Hv1 associated disease or condition. In some embodiments, a subject is considered to be suffering from an Hv1 associated disease or condition if the subject is displaying one or more symptoms commonly associated with said disease or condition. Synthetic peptides as provided herein, or Hv1 modulating agent compositions, may be administered prior to or after development of one or more such symptoms. For example, synthetic peptides as provided herein, or Hv1 modulating agents, are used to ameliorate inflammation, allergies, autoimmunity, cancer, asthma, brain damage from ischemic stroke, Alzheimer's disease, infertility, and numerous other conditions. In some embodiments, the desired outcome is reduction in intensity, severity, and/or frequency, and/or delay of onset of one or more of these conditions. Additionally or alternatively, Hv1 modulating agents may be used as a form of birth control by blocking sperm function.
In alternative embodiments, pharmaceutical compositions as provided herein comprise a synthetic peptide as provided herein, for example, Hv1 modulating agents, used to change any of the functions of Hv1 channels described in the present disclosure to achieve a preferred or therapeutic outcome. As described herein, Hv1 channels transport protons across cell membranes and are expressed in a variety of cells and tissues. Functions of Hv1 channels differ depending on the cells in which they are expressed. Uses for Hv1 modulating agents can include increasing or decreasing proton current across cell membranes and/or increasing or decreasing pH in the cytosolic, extracellular, or intraluminal space of cells.
In some embodiments, uses for synthetic peptides as provided herein, for example, Hv1 modulating agents, include effects on Hv1-related processes. For example, in some embodiments, Hv1 modulating agents may be used to increase or decrease the expression and/or function of NOX enzymes, including NOX1, NOX2, NOX3, and/or NOX4. In some embodiments, Hv1 modulating agents may be used to increase or decrease production of ROS.
In some embodiments, uses for synthetic peptides as provided herein, for example, Hv1 modulating agents, include altering biological functions in specific cells. For example, the function of Hv1 channels in white blood cells includes extrusion of protons to facilitate ROS production via NOX activity in the phagosome. This process allows white blood cells to destroy bacteria and other pathogens. In some embodiments, uses of Hv1 modulating agents may include changing these functions in white blood cells. Alternatively, Hv1 channel function in human sperm has been associated with sperm capacitation, activation and mobility to achieve fertilization. In some embodiments, Hv1 modulating agent uses include increasing or decreasing sperm function and/or fertilization ability.
Products of Manufacture and KitsProvided are products of manufacture and kits for practicing methods as provided herein; and optionally, products of manufacture and kits can further comprise instructions for practicing methods as provided herein.
In addition to the synthetic peptide as provided herein, for example, Hv1 modulating agent, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer or a preservative, and/or a second agent for treating a condition or disorder described herein. Alternatively, other ingredients can be included in the kit, but in different compositions or containers than the Hv1 modulating agent. In such embodiments, the kit can include instructions for admixing the Hv1 modulating agent and the other ingredients, or for using the Hv1 modulating agent together with the other ingredients.
Alternatively or additionally, contents of kits may include, but are not limited to, expression plasmids containing nucleotides (or characteristic or biologically active portions) encoding synthetic peptide as provided herein, for example, Hv1 modulating agents (or characteristic or biologically active portions or fragments thereof). Alternatively or additionally, kits may contain expression plasmids that express synthetic peptide as provided herein, for example, Hv1 modulating agents (or characteristic or biologically active fragments or portions thereof). Alternatively or additionally, kits may contain isolated and stored synthetic peptide as provided herein, for example, Hv1 modulating agents.
In certain embodiments, kits for use as provided herein include, a reference sample, instructions for processing samples, performing tests on samples, instructions for interpreting the results, buffers and/or other reagents necessary for performing tests. In certain embodiments the kit can comprise a panel of antibodies.
Provided are kits for administration of pharmaceutical compositions. For example, in some embodiments, provided are kits comprising at least one dose of an Hv1 modulating agent. In some embodiments, the invention provides a kit comprising an initial unit dose and one or more subsequent unit doses of an Hv1 modulating agent. In some such embodiments, the initial unit dose is greater than the subsequent unit doses or wherein all of the doses are equal.
Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed here in the Summary, Figures and/or Detailed Description sections.
As used in this specification and the claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About (use of the term “about”) can be understood as within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12% 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Unless specifically stated or obvious from context, as used herein, the terms “substantially all”, “substantially most of”, “substantially all of” or “majority of” encompass at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, or more of a referenced amount of a composition.
The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Incorporation by reference of these documents, standing alone, should not be construed as an assertion or admission that any portion of the contents of any document is considered to be essential material for satisfying any national or regional statutory disclosure requirement for patent applications. Notwithstanding, the right is reserved for relying upon any of such documents, where appropriate, for providing material deemed essential to the claimed subject matter by an examining authority or court.
Modifications may be made to the foregoing without departing from the basic aspects of the invention. Although the invention has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, and yet these modifications and improvements are within the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. Thus, the terms and expressions which have been employed are used as terms of description and not of limitation, equivalents of the features shown and described, or portions thereof, are not excluded, and it is recognized that various modifications are possible within the scope of the invention. Embodiments of the invention are set forth in the following claims.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
EXAMPLESUnless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
Example 1: Human Sperm and Neutrophils Require Direct Activation of the Proton Channel by Albumin to Operate/Direct Activation of the Proton Channel by Albumin Leads to Human Sperm Capacitation and Sustained Release of Inflammatory Mediators by NeutrophilsThis example demonstrates that methods and compositions as provided herein are effective for inhibiting, ameliorating or preventing PMN-mediated inflammation in vivo.
Albumin (Alb) is shown here to activate human voltage-gated proton channels (hHv1). Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the high level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Similarly, Alb activation of hHv1 in neutrophils allows sustained production and release of reactive oxygen species and proteases upon immune stimulation. One Alb binds to the two hHv1 voltage sensor domains (VSDs), enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that bind the two hHv1 VSDs and suggests an electrostatic gating modification mechanism that favors their active “up” conformation. This report resolves longstanding conundrums: how hHv1 opens at negative sperm membrane potentials and why Alb facilitates in vitro fertilization, and describes new cellular physiology that is widespread.
Regulation of intracellular pH (pHi) is essential to cell biology in health and disease1 and, since its identification2, 3, the human voltage-gated proton channels (hHv1) has been recognized to be both widespread and central in many of these processes4. Recently, we used a designed peptide inhibitor (C6) of the channel to demonstrate that H+ efflux via hHv1 is required in human sperm to induce intracellular alkalization and initiate capacitation, and essential in human neutrophils to maintain cytoplasmic pH during the respiratory burst to allow reactive oxygen species (ROS) production5, in support of earlier proposals6-10.
hHv1 channels are comprised of two identical subunits, each with four transmembrane spans (TMs) that resemble the voltage sensor domains (VSDs) in other voltage-gated ion channels (VGICs) but lack the two additional TMs that contribute to forming the ion conduction pores in those channels2, 3. In hHv1, there are two H+-selective conduction pathways, one in each subunit11-13.
Albumin (Alb), is the most abundant protein in interstitial fluids where it is present at various levels (7-30 mg/mL)14 and human plasma (34-54 mg/mL) where it is recognized to transport hormones, metabolites and drugs, serves as a circulating antioxidant, and supports oncotic pressure15. This ubiquitous globular protein is composed of 585 amino acids and folds into three domains16.
We wondered about a physiological connection between Alb and hHv1 based on its contrasting concentrations in semen (just 1 mg/mL, 15 μM)17 and in uterine fluid (34 mg/mL, 500 μM)18 and the documented increase in reproductive success when in vitro fertilization (IVF) solutions are supplemented with Alb19, 20 Furthermore, Alb is implicated in deleterious systemic inflammatory responses mediated by neutrophils after cardiopulmonary bypass and in periodontal disease21, 22.
Here, we demonstrate that Alb binds directly to hHv1 to activate the channel, increasing the open probability and H+ current. In human sperm, this initiates capacitation while in human neutrophils it increases peak levels of ROS release, sustains ROS production during the respiratory burst, and stimulates release of proteases in response to immune stimulation. The stoichiometry of binding, inferred by the Hill coefficient for changes in H+ currents in response to Alb dose, is confirmed to be one Alb per channel using single molecule total internal reflection fluorescent (smTIRF) microscopy. Alb is shown to bind to hHv1 on the external residues linking the third and fourth transmembrane segments that comprise the voltage sensor (S3-S4 loop) in each subunit using point mutations and chimeric channels generated between hHv1 and the proton channel from Ciona intestinalis, CiHv13. Modeling with molecular dynamics (MD) simulation points to two binding sites formed by residues in Alb domain II (DII) for the two S3-S4 loops in each hHv1 channel. The two Alb sites are validated by the effects of mutagenesis on binding as assessed by fluorescence resonance energy transfer (FRET) microscopy and electrophysiology. The structural model of the Alb-hHv1 macromolecular complex suggests an electrostatic gating modification mechanism through which binding of Alb facilitates opening of hHv1 channels by favoring the active “up” conformation of the two VSDs. The essential stimulatory role of Alb in the physiology of both sperm and neutrophils via hHv1 suggests that Alb will have as-yet unrecognized roles in the many other tissues where the channel is critical, including, the heart, the central nervous system, and cancers of the breast and gastrointestinal tract23, 24.
ResultsAlb Acts on hHv1 in Human Sperm to Initiate Capacitation
Human sperm undergo a process called capacitation in the female reproductive tract25, whereby pHi rises stimulating Ca2+ influx and mobility changes26. This process endows sperm with the capacity to undergo the exocytotic acrosomal reaction required to penetrate the zona pellucida and fertilize the oocyte27. Here, Alb was observed to activate hHv1 channels in live human sperm by recording proton currents in mature non-capacitated spermatozoa, using whole-cell patch clamp as previously described5.
The native proton channel currents were elicited by depolarizing voltage steps of 1.5 s from a holding potential of −60 mV every 10 s with a approximately 30-fold proton gradient (pHi=6.0 and pHo=7.4). When 80 μM Alb was applied to the sperm, the currents increased approximately 3-fold, accompanied by a approximately 3-fold acceleration of activation and a approximately 1.6-fold slowing of deactivation at +60 mV (
We next focused on the effect of Alb on pHi, as capacitation first requires H+ efflux via hHv1 to alkalinize sperms5-7. The fluorescent ratiometric pH probe BCECF was used to evaluate changes in the cytosolic pH, as previously described28. As anticipated, 75 μM Alb induced a robust increase in pHi in non-capacitated human sperm when compared to a control Fab protein or after blocking hHv1 with C6 (
To confirm that Alb-induced changes in pHi increased Ca2+ influx through CatSper5-7, non-capacitated sperm incubated with Alb were exposed to progesterone, a stimulus that triggers the acrosome reaction only if sperm have been capacitated by changes in pHi and intracellular calcium levels ([Ca2+],)29, 30. In a concentration dependent manner, Alb enhanced progesterone-induced increases in [Ca2+]i, a change that was suppressed by the hHv1 blocker C6 (
The final step, the exocytotic acrosomal reaction, requires prior capacitation30 and was rarely observed (approximately 2%) when we added progesterone to non-capacitated sperm in the absence of Alb (
Alb Stimulates hHv1 in Neutrophils to Augment ROS Production and Elastase Release
Human neutrophils undergo a respiratory burst to produce ROS as a principal effector mechanism to kill bacteria32. During the respiratory burst, the NADPH oxidase 2 (NOX2) transfers electrons across the membrane, resulting in membrane depolarization and cytoplasmic acidification that suppresses ROS production33. To sustain NOX2 activity and ROS production, H+ efflux is required to maintain physiological pHi and membrane potential23, 34.
Here, activation of hHv1 in neutrophils by Alb was demonstrated first by recording native proton currents in freshly-isolated cells from human peripheral blood by whole-cell patch clamp. Application of 450 μM Alb led to a approximately 2.5-fold increase in native proton currents at +60 mV, with a approximately 6-fold acceleration of activation and a approximately 1.5-fold slowing of deactivation, offering an estimated equilibrium affinity (Kd) of 112±9 μM (
To assess ROS release from the neutrophils, we used a luminol-amplified chemiluminescence assay for superoxide anion (O2′−) measurement35. Formylated bacterial peptides like fMLF stimulate the production of ROS by neutrophils by binding to G-protein coupled receptors36 and triggering intracellular pathways that induce NOX2 to produce O2′−4, 37. Here, we observed that 1 μM fMLF stimulated a transient rise and decay in ROS release from neutrophils as expected, whereas the additional presence of 450 μM Alb led to an increase in the peak magnitude of ROS release. More significantly, Alb allowed for sustained ROS production that was still elevated at 60 min, long after release had returned to baseline in the absence of Alb (
ROS release potentiated by Alb was concentration-dependent across the physiological range, becoming apparent at 15 μM and maintained at a approximately 7-fold increased rate of release after 60 min with 450 μM (
During the inflammatory response, neutrophils also release antimicrobial proteases that cleave bacterial virulence factors, such as elastase38. Thus, fMLF-induced neutrophil degranulation can be quantified by measurement of elastase release21.
hHv1 shows an “enhanced gating mode” during the neutrophil respiratory burst increasing the likelihood of channel opening that is reported to reflect protein kinase C (PKC) phosphorylation of Thr29 at N-terminus of hHv139. Indicating that Alb-activation was not mediated by this mechanism, we observed that hHv1-T29A channels, which are not subject to PKC phosphorylation, were activated by Alb like WT channels (Supplementary
Alb Also Activates Cloned hHv1 by Changing the Voltage-Dependence of Gating
To explore the mechanism of Alb activation, hHv1 channels were expressed in HEK293T cells and studied by whole-cell patch clamp with a 10-fold proton gradient (pHi=6.5 and pHo=7.5), as previously described5. In the absence of Alb, hHv1 currents activated slowly in response to depolarization and showed fast deactivation (
Alb stimulation of hHv1 currents was reversible and concentration-dependent with association and dissociation constants of kon=1.3×103±0.1×103 M−1s−1 and koff=0.084±0.009 s−1 determined by single-exponential fits to the time courses for activation and deactivation, respectively (
The designed peptide C6 inhibits hHv1 by binding to the S3-S4 loops with positive cooperativity, one peptide on each loop (
This effect of Alb showed specificity and required that the protein was intact, as neither Fab nor Alb subjected to Proteinase K digestion activated hHv1 at concentrations up to 800 μM (Supplementary
A recent report indicates that a protease-digested form of hHv1 (Hv1Sper) with 68 residues removed from the N-terminus represents as much as half the channel protein isolated from human sperm when visualized by western blotting40. We constructed the foreshortened channel by genetic deletion of the residues, expressed it in HEK293T cells and observed the approximately −30 mV shift in the G-V relationship compared to WT hHv1 reported by Berger and colleagues40. Indicating that the truncated channels are also sensitive to Alb regulation, we observed application of 500 μM Alb to accelerate activation, slow deactivation, increase current magnitude, and to shift the G-V curve by −15 mV (Supplementary table 1 and supplementary
Because Human Tubal Fluid Medium (HTF, pH 7.2) is routinely used for human IVF41 to mimic the native environment, we sought to confirm that the effect of Alb on hHv1 channels was apparent in this solution using a pipette solution at pH 6.7 as estimated for sperm in the human uterus42. Indeed, under these conditions, 500 μM Alb increased hHv1 current approximately 10-fold at 0 mV, shifting the V1/2 for conduction by −40 mV (Supplementary table 1 and Supplementary
Alb stimulates hHv1 in neutrophils to augment ROS production and elastase release Human neutrophils manifest a respiratory burst to produce reactive oxygen species (ROS) as a principal effector mechanism to kill bacteria (El-Benna et al., 2016; Winterbourn et al., 2016). During the respiratory burst, the NADPH oxidase complex (NOX2) transfers electrons across the membrane, resulting in membrane depolarization and cytoplasmic acidification that suppresses ROS production (DeCoursey et al., 2003). In order to sustain NOX2 activity and ROS production, H+ efflux is required to maintain physiological pHi and membrane potential (DeCoursey, 2004; Seredenina et al., 2015).
Here, activation of hHv1 in neutrophils by Alb was demonstrated first by recording native proton currents in freshly-isolated cells from human peripheral blood by whole-cell patch clamp. Application of 450 μM Alb led to an approximately 2.5-fold increase in native proton currents at +60 mV, with an approximately 6-fold acceleration of activation and an approximately 1.5-fold slowing of deactivation, offering an estimated Kd of 112±9 μM (
To assess ROS release from the neutrophils, we used a luminol-amplified chemiluminescence assay for superoxide anion (O2′−) measurement (Bedouhene et al., 2017). Formylated bacterial peptides like fMLP stimulate the production of ROS by neutrophils (Iizawa et al., 1995) by binding to G-protein coupled receptors (Wittmann et al., 2002) and triggering intracellular pathways that induce NOX2 to produce O2′− (DeCoursey, 2013; Worthen et al., 1994). Here, we observed that 1 μM fMLP stimulated a transient rise and decay in ROS release from neutrophils as expected, whereas the additional presence of 450 μM Alb led to an increase in the peak magnitude of ROS release. More significantly, this allowed for sustained ROS production that was still elevated at 60 min, long after release had returned to baseline in the absence of Alb (
ROS release potentiated by Alb was concentration-dependent across the physiological range, becoming apparent at 15 μM and maintained at an approximately 7-fold increased rate of release after 60 min with 450 μM (
During the inflammatory response, neutrophils also release antimicrobial proteases that cleave bacterial virulence factors, such as elastase (Amulic et al., 2012). Thus, fMLP-induced neutrophil degranulation can be quantified by measurement of elastase release (Rabaglia et al., 2002).
Alb Binds to the External S3-S4 Loop of hHv1
We looked for Alb binding sites on the extracellular portions of hHv1, that is, the residues linking the four membrane-spanning segments, the S1-S2 loop and the S3-S4 loop (
CiHv1 channels carrying both the S1-S2 and S3-S4 loops of hHv1 (hS1-S2, S3-S4CiHv1) were endowed with sensitivity to Alb, whereas transplanting only the S1-S2 loop from hHv1 into CiHv1 (hS1-S2CiHv1) was insufficient to confer Alb-activation (
To explore the role of the hHv1 S3-S4 loop in Alb binding, we carried out a mutational scan where loop residues 1183 to L204 were changed individually to Cys and the response of the mutant channels to Alb was studied (
One Alb Binds to One Dimeric hHv1 Channel
The increased affinity of Alb for hHv1-G199L channels allowed a direct study of binding using smTIRF and live cells (Methods). Alb was labeled with carboxytetramethylrhodamine (TAMRA) on its N-terminus to yield TAMRA-Alb and the channel was tagged on its C-terminus with a teal fluorescent protein to produce hHv1-G199L-TFP (
Control experiments with hHv1-G199L-TFP subunits showed two stepwise decreases in fluorescence in 89% of particles (
When 3 μM TAMRA-Alb was applied to cells expressing hHv1-G199L-TFP, smTIRF microscopy yielded a mean Manders' coefficient for colocalization (MCC) of 0.50±0.06, suggesting that roughly half the channels were associated with TAMRA-Alb (Supplementary Table 3). Of the hHv1-G199L-TFP particles directly confirmed to be intact dimeric channels (that is, showing two TFP bleaching steps) and to be colocalized with TAMRA-Alb, 95% were observed to be associated with one Alb, and 5% were visualized with two (
As an independent test of the 1 Alb to 1 dimeric hHv1 channel stoichiometry, we studied monomeric hHv1 (ΔhHv1), engineered by shortening the N-terminus and removing the C-terminus coiled-coil domain, as described5. Supplementary
Alb DII is the Principal Domain for Binding to hHv1
To facilitate characterization of the Alb-hHv1 interaction by mutational screening, we constructed a gene allowing expression of Alb variants on the extracellular surface of cells via a membrane tether. The construct was comprised by the nucleotides encoding Alb (or Alb variants) in-frame with the code for the transmembrane helix of the platelet-derived growth factor receptor carrying the fluorescent protein mVenus (VFP) on its intracellular C-terminus to produce T-Alb-VFP (Methods)44. This permitted study of the interaction of T-Alb-VFP constructs and hHv1 tagged with teal fluorescent protein on its C-terminus (hHv1-TFP) in live cells (
When T-Alb-VFP was expressed with hHv1-TFP, it potentiated the channel to a similar extent as soluble Alb. T-Alb-VFP increased current density at 0 mV approximately 3-fold, speeding the kinetics of activation approximately 4-fold, slowing deactivation approximately 2-fold, and shifting V1/2 by −12 mV, values consistent with an effective concentration of free Alb in solution of approximately 35 μM (
The physical interaction of T-Alb-VFP with hHv1-TFP at the membrane surface of HEK293T cells was measured using the photobleaching rate of the TFP donor (
Alb consists of three homologous domains: DI (residues D1 to R197); DII (residues L198 to Q385), and DIII (residues N386 to L585)15 47 We investigated how each domain contributed to hHv1 binding by developing six tethered Alb domain variants, T-DI-VFP; T-DII-VFP; T-DIII-VFP; T-DI-DII-VFP; T-DII-DIII-VFP; and T-DI-DIII-VFP (Methods). When the interaction between tethered Alb domains and hHv1 channels on the cell surface were measured by FRET, the variant consisting Alb DII alone (T-DII-VFP) interacted with hHv1-TFP like WT Alb (T-Alb-VFP) (
In the absence of high resolution information for the Alb-hHv1 complex, we performed an in silico docking analysis (HPEPDOCK™)48 using the crystal structure of Alb (PDB 1BM0)16 and the 11-residue binding epitope in S3-S4 loop of hHv1 (F190 to L200). Initial docking predicted two sites in Alb, separated by approximately 27 Å, as putative hot spots for interaction (Supplementary
We tested the docking predictions by generating eleven T-Alb-VFP variants, three in Site 1 (E188A, H288A, E292A) with nearby E184A as a control, and six in Site 2 (Y334A, R337A, H338A, D340A, F374A, V381A) with nearby E311A as a control. Each T-Alb-VFP mutant was evaluated for its impact on the interaction with hHv1-TFP in live cells using FRET (
Model of Alb Binding to hHv1
A structural model of the Alb-hHv1 complex was generated from MD simulations using the NAMD™ program49, based on our knowledge of the interfacial residues identified as critical for binding by patch-clamp (
The final Alb-hHv1 model reveals specific interactions (
hHv1-L200 was found to influence Alb activation (
Two experiments inspired by the MD simulation results were performed and further supported the model of the Alb-hHv1 complex. First, an unanticipated hydrogen bond between Alb-E382 near Site 2 and hHv1-Y134 (subunit B) was observed in the model. When the Glu residue was neutralized by mutation to alanine (producing T-Alb-E382A-VFP), the binding affinity of Alb to hHv1-TFP assessed by FRET was reduced (
Consistent with the notion that Alb binds to the hHv1 VSDs to activate the channels, the binding affinity of Alb was observed to increase with membrane depolarization as judged by FRET. Thus, the proton channel VSDs move “outward” to the active conformation in response to increased membrane potential4′ 5 and we observe that the affinity of the interaction of T-Alb-VFP and hHv1-TFP increased as the resting membrane potential (RMP) was increasingly depolarized from −43±6 mV to −7±2 mV by isotonic replacement of NaCl with KCl in the bath solution (
Two outstanding questions in sperm physiology are answered by our identification of Alb activation hHv1 channels as central to initiation of capacitation. First, the RMP of non-capacitated sperm is estimated to be −20 mV to −40 mV51, whereas the threshold for opening of hHv1 channels was reported to be approximately 0 mV in the ionic environment present in the uterus4, 52, 53. This presented an enigma, how did hHv1 open under natural conditions? Here, we answer to the conundrum; because the uterus contains approximately 500 μM Alb, the Vthreshold for hHv1 shifts to approximately −30 mV (Supplementary
Alb has been proposed to extract cholesterol from the sperm plasma membrane thereby allowing HCO3− influx to alkalinize the cells54, apparently obviating a role for H+ flux via hHv1 that we demonstrated using C6 to block the channel5. Here, we observe that when Alb is pre-saturated with cholesterol it induces the same increase in pHi in non-capacitated human sperm as Alb alone (Supplementary
A second hypothesis for augmented fertilization success with Alb has been that it sequesters Zn2+, decreasing the already low concentration of Zn2+ in the uterus compared to semen57, thereby releasing hHv1 from residual Zn2+ inhibition?. While such a role for Alb remains possible, we observe that Alb has similar stimulatory effects on hHv1-H140A and WT hHv1 channels (Supplementary
Furthermore, hHv1 activation by Alb does not appear to result from chelation of trace elements. It has become commonplace to add EDTA or EGTA to bath solutions for biophysical studies of hHv1 because this speeds current activation, slows deactivation, and shifts the G-V relationships to more hyperpolarized potentials, increasing the current magnitude so the channel is easier to study4. The effects of EDTA and EGTA have been attributed to chelation of trace heavy metals contaminating salt solutions4. We do not support the notion that Alb activates hHv1 by chelation of trace metals, first, because Alb activation is increased (or decreased) by mutating sites on the channel (
Similar to what we observed in sperm, 500 μM Alb alone shifts the V1/2 of cloned hHv1 channels in HEK293T cells −45 mV (Supplementary table 5). However, in contrast to sperm, EDTA and EGTA equally effective as Alb on the heterologously-expressed channel, inducing a shift approximately −40 mV. Of note, the interaction of T-Alb-VFP and hHv1-TFP measured by FRET increases on application of 1 mM EGTA, in the same fashion as membrane depolarization (
Alb potentiation of hHv1 is established here as a required and natural mechanism for capacitation initiation by three observations (
Alb Modulates the Innate Immune System Via hHv1
Neutrophils are the most abundant white blood cell type and an essential part of the innate immune system that defends against invading pathogens via ROS release, a process that is also implicated in pathogenesis related to oxidative stress32. When stimulated, neutrophil NOX2 produces ROS by reducing O2 to O2*, a reaction that translocates electrons across the plasma membrane, depolarizing cells, and generating protons in the cytoplasm that inhibit further NOX2 activity23, 33. Proton efflux via Hv1 allows sustained ROS production by NOX2 by maintaining appropriate membrane potential and pHi8, 10, as demonstrated by genetic deletion of Hv1 in mice9 and by C6 blockade in human neutrophils5.
In this study, we used fMLF to induce the respiratory burst in human neutrophils and show that Alb acts via hHv1 to enhance NOX2 activity. Indeed, Alb was previously observed to increase ROS production by neutrophils although the relevant receptor and mechanism were unknown22. The importance of Alb potentiation of hHv1 in neutrophils can be inferred from estimates that during the respiratory burst the cells would depolarize from roughly −60 mV58 to +200 mV within 20 ms without charge compensation by hHv134, and that activation of hHv1 is slower in the absence of Alb (
A number of the attributes of the neutrophil response appear to protect against aberrant inflammatory mediator release. Alb in the absence of a pathogenic stimulus does not induce ROS production (Supplementary
A Physical Model for Alb Binding on hHv1
Given the critical physiological roles demonstrated for Alb via hHv1, and in the absence of high resolution atomic structural information, we judged it valuable and feasible to construct a structural model of the Alb-hHv1 macromolecular complex. The Alb-hHv1 model was constructed by MD simulations (
Some neurotoxins target VGICs to activate or inhibit the channels by favoring the “up” or “down” state of the voltage sensors, respectively. By this mechanism CssIV activates Nav1.2, while C6 inhibits hHv15, 62. We suggest that the physiological effects of Alb on hHv1 take place by a similar mechanism, whereby Alb favors the activated position of the two hHv1 voltage sensors. hHv1 responds to membrane potential changes via three arginine residues, R205, R208 and R211 in the S4 helix (
Supporting the notion that Alb acts as a gating modifier, it shifts the activation of hHv1 to more negative potentials (
hHv1 is implicated in a wide range of biological processes in addition to the capacitation of sperm and the innate immune responses we study here4. The channels have some notable roles in proliferation of cancer cells24, tissue damage during ischemic stroke64, and hypertensive injury of the kidney65. Because Alb is ubiquitous at levels that vary in different human compartments in health and disease14, the potentiation of hHv1 by Alb we describe in this report will be wide-spread, tissue-dependent, and play both salutary and unfavorable roles in human physiology.
Method Details Molecular BiologyThe coding sequences for human Hv1 (NM_001040107) or Ciona intestinalis Hv1 (NM_001078469) were tagged with a teal fluorescent protein (TFP) via a 13-residue linker using GBLOCK™ gene fragments (Integrated DNA Technologies) and inserted into a laboratory dual-purpose vector pMAX using Gibson Assembly (New England BioLabs). hS1-S2, S3-S4CiHv1 was constructed by replacing the nucleotides for the S1-S2 loop (L169-L187) and S3-S4 loop (G231-L254) of CiHv1 with S1-S2 loop (I121-F139) and S3-S4 loop (I183-L204) of hHv1. Similarly, hS1-S2CiHv1 was constructed by replacing the S1-S2 loop (L169-L187) of CiHv1 with S1-S2 loop (I121-F139) of hHv1; hS3-S4CiHv1 was constructed by replacing the S3-S4 loop (G231-L254) of CiHv1 with S3-S4 loop (I183-L204) of hHv1; and, CiS3-S4Hv1 was constructed by replacing the S3-S4 loop (I183-L204) of hHv1 with S3-S4 loop (G231-L254) of CiHv1. All chimeras were assembled into pMAX using Gibson assembly. Hv1Sper was constructed by truncating 67 residues at the N-terminus of hHv1 (residues from A2 to R68). hKv1.3 (NM_002232), hKv1.5 (NC_000012), hKv2.1 (NP_004966), hKCNQ1 (NM_000218), hKCNE1 (NP_000210.2), hNav1.5 (NC_000003) and the sodium voltage-gated channel R subunit 1 (NC_000019) in pMAX were used as previously described. Tethered Albumin (Alb) constructs were built in pMAX vector. To generate T-Alb-VFP, the PDGF-receptor transmembrane domain was amplified and inserted into pMAX vector. Subsequently, sequences encoding the preprotrypsin secretory pathway signal sequence (secretion signal), Alb sequence and a 16 residues flexible linker region (Gly-Asn)×8 were inserted at the 5′ end of the PDGF transmembrane domain. Fluorescent protein mVenus (VFP) was attached to the C-terminus of transmembrane domain. T-C6-VFP with C6 sequence replacing Alb and T-NO-VFP without Alb sequence incorporation was constructed using the same strategy. Tethered Alb domain constructs with sequence insertion of DI (residues 1-194), DII (residues 183-387), DIII (residues 381-585), DI-DII (residues 1-387), DII-DIII (residues 183-585), and DI-DIII (residues 1-194 and 381-585, respectively) were generated47 using PCR and then ligated into pMAX vector. Point mutations for hHv1 and T-Alb-VFP were introduced using QuikChange Site-Directed Mutagenesis Kit (Agilent). The sequences of all constructs were confirmed by DNA sequencing. Because hHv1 with V187C, L189C and L203C changes did not have expression in HEK293T cells, alanine mutations were studied at these three sites.
Cell CultureHEK293T cells (RRID: CVCL_0063) were obtained from ATCC and used for heterologous expression. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (ATCC) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Gibco) and incubated at 37° C. in a humidified atmosphere containing 5% CO2/95% air. Plasmids were transfected into cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Experiments were preformed 24-48 hours post transfection.
Proteins, Peptides and ReagentsC6 toxin (MH828728) was purchased as synthetic peptides from CSBio. Peptide toxin folding reactions were quenched by acidification and purified by reverse-phase HPLC, as before5. Peptides that were more than 95% pure were lyophilized and stored at −20° C. The composition of the peptides was confirmed by mass spectral analysis. Peptides were dissolved in appropriate external solutions for whole-cell patch clamp recordings or physiological assays before use. Albumin from human serum (lyophilized powder, fatty acid free) was purchased from Sigma (A1887). Proteinase K (Lyophilized) was purchased from Promega (V3021) and the digestion was performed following the protocol supplied with the product. Purified Fab fragment of human IgG was purchased from GenWay Biotech (GWB-DD0665). Alb was labeled with 5,6-TAMRA-SE (5-(and-6-)-carboxytetramethyl-rhodamine succinimidyl ester) (Life Technologies) per manufacturer instructions. Thus, Alb was dissolved in 50 mM HEPES, 100 mM NaCl at pH 7.5 to 10 mg/mL concentration. 5,6-TAMRA-SE dye was dissolved in 500 μL DMSO. Alb and dye were mixed at a molar ratio of 1:10 for 1 h at room temperature on a rotating shaker. The labeled Alb was purified by HPLC over a 20-80% acetonitrile gradient and the corresponding single peak was collected. Samples were lyophilized and stored at −80° C. Labelled protein was dissolved in appropriate external solution before use. All the reagents and chemicals were purchased from Sigma-Aldrich unless otherwise stated.
Human Sperm PreparationEjaculates were obtained from healthy donors by masturbation after at least 48 h of sexual abstinence. Only semen samples that fulfilled the World Health Organization (WHO 2010) guidelines were selected for experiments. All semen donors gave written informed consent and the protocol for semen sample handling were approved by the Ethic Committee of the School of Medicine, National University of Cuyo and the Bioethics Committee at the Biotechnology Institute from the National Autonomous University of Mexico. After sample liquefaction (30 minutes at 37° C.), motile sperm were recovered after a swim-up separation for 1 h at 37° C. as described previously5.
Human Peripheral Blood Neutrophils PurificationHuman polymorphonuclear neutrophils were isolated from peripheral blood from healthy donors by Ficoll-Paque Plus (GE Healthcare) density-gradient centrifugation. Peripheral blood was obtained from Institute for Clinical and Translational Science of University of California Irvine. Donor population is composed of 50% female and 50% male with ages ranging from 23-62 years old. 20 mL blood was mixed with 3% dextran in PBS (Sigma-Aldrich) for 20 min in a 50 mL conical tube. The top clear layer containing leukocytes was collected and underlaid with 10 mL of Ficoll-Paque Plus. The cell suspension was centrifuged at 500 g for 30 min at 20° C. to separate mononuclear cells from neutrophils and the remaining red blood cells. The overlying plasma and monocyte layers were aspirated, and the neutrophils and red blood cells pellet was re-suspended in Red Blood Cells Lysis Buffer (eBioscience), incubated for 10 min to lyse red blood cells. 35 mL PBS was added to stop the lysis and the cell suspension was centrifuged at 300 g for 5 min at 4° C. Cell pellet was re-suspended in RPMI1640 (Gibco). An aliquot of neutrophils was mixed with Trypan blue (Gibco) and counted using a hemocytometer. Neutrophils isolated using this method were routinely found to be greater than 97% of the final cell preparation.
ElectrophysiologyProton current from hHv1, CiHv1 and chimera channels were recorded in whole-cell mode using an Axopatch 200B amplifier. Stimulation and data collection were achieved with a Digidata1440A and pCLAMP 10 software (Molecular Devices). HEK293T cells expressing hHv1 channel variants were perfused with a nominally divalent cation-free external solution of 100 mM HEPES, 100 mM NaCl, 10 mM glucose at pH 7.5 or human tubular fluid medium (HTF) comprising 101.6 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2), 25 mM NaHCO3, 2.78 mM Glucose, 0.33 mM Na pyruvate and 21.4 mM Na lactate at pH 7.2, unless otherwise noted. Pipettes with resistances between 3-5 MΩ were filled with 100 mM Bis-Tris buffer, 100 mM NaCl, and 10 mM glucose at pH 6.5 or 6.7. For EGTA recordings, 1 mM EGTA was added in the external solution with the supplement of 2 mM MgCl2. Sampling frequency was 10 kHz and was filtered at 1 kHz. Alb was applied in the external solution through a multichannel micro-perfusion system after currents monitored by test pulses to 0 mV for 1.5 s from a holding voltage of −60 mV, with 10 s interpulse intervals, were judged to be stable. Current-voltage relationships were evoked from a holding potential of −60 mV to test pulses from −60 mV to +60 mV for 1.5 s in 20 mV intervals every 10 s.
Proton currents in human neutrophils were recorded with an external solution of 100 mM HEPES, 130 mM NMDG, 10 mM glucose at pH 7.5. Pipettes with resistances between 10-15 MΩ were filled with 100 mM MES buffer, 130 mM NMDG, and 1 mM EGTA at pH 6.0. Current-voltage relationships were evoked from a holding potential of −60 mV to test pulses from −60 mV to +60 mV for 5 s in 20 mV intervals every 15 s. Current was assessed at the end of the test pulse.
The G-V relationships were determined as described by DeCoursey68, the reversal potential (Vrev) is calculated with the equation Vrev=(Iend−Itail)/(Vtest−Vhold), and were fit to the Boltzmann equation, G=Gmax/[1+exp(−zF(V−V1/2)/RT)], where Gmax is maximum conductance, V is the test potential, V1/2 is the voltage of half-maximal activation, z is the effective valence, T is the temperature, R is the gas constant, and F is the Faraday constant. Deactivation kinetics for hHv1 with and without Alb were determined by fitting traces with single exponential functions. Activation kinetics were fit with a single exponential having a delay. kon and koff were estimated from fits of the kinetics of toxin wash-in and wash-out and calculated using equations:
A dose-response curve was determined by normalizing the current or the time constants of channel activation (tact) to the values before the application of Alb, then plotting versus concentration of Alb. Dose-response curves were fitted with a Hill equation (Eqn 3) in Origin 8.0.
where r is the rate of hHv1 current increasing with Alb at equilibrium, [Alb] is the concentration of Alb, and h is the Hill coefficient. The equilibrium affinity (Kd) of Alb for hHv1 binding was estimated similarly by Hill equation, using the rate of hHv1 τ-act increasing and assuming Hill coefficient is 1 (simple bimolecular interaction). Perforated patch clamp was performed with nystatin at 150 pg/ml in the pipette solution. After seal formation, the resting membrane potential of cells expressing hHv1 channels was measured in current-clamp configuration after attainment of whole-cell configuration with 10 mM HEPES, 136 mM KCl, 1 mM MgCl2, 2 mM K2ATP, 5 mM EGTA, pH 7.2 in the pipette and the bath solution described in the section Live cell FRET microscopy.
Xenopus laevis oocytes were injected with cRNA encoding hHv1 and proton current was measured using two-electrode voltage clamp (TEVC) three days thereafter.
Recording solution for hHv1 was 60 mM NaCl, 1 mM MgCl2, 2 mM CaCl2), 120 mM HEPES, pH 7.3. Before hHv1 recording, we injected oocytes with 50 nL of 1 M HEPES (pH=7.3) to minimize pH changes due to proton efflux. This results in approximately 100 mM HEPES in the cytosol. For recording the potassium currents, Xenopus laevis oocytes were injected with cRNA encoding hKv2.1 or hS3-S4Kv2.1.
Recording solution was composed of 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 0.3 mM CaCl2), 10 mM HEPES, pH 7.5. Recordings were performed with constant gravity flow of solution at 2 mL/min yielding chamber exchange in approximately 5 s. Currents were recorded 1-2 days after cRNA injection using an Oocyte clamp amplifier OC-725C (Warner Instruments, Hamden, CT), and electrodes filled with 3 M KCl with resistance of 0.3-1 MΩ. Data were filtered at 1 kHz and digitized at 20 kHz using pCLAMP 10 and assessed with Clampfit 10 and Origin 8.
Procedures of human sperm electrophysiology were approved by the Bioethics Committee at the Biotechnology Institute from the National Autonomous University of Mexico. Motile sperm were recovered after a swim-up separation for 1 h in modified Krebs-Ringer bicarbonate medium under non-capacitation conditions (without BSA and Ca2+) at 37° C. in a humidified atmosphere of 5% CO2-95% air. Spermatozoa were stored in physiological solution comprising 135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2), 5 mM glucose, 1 mM sodium pyruvate, 10 mM lactose and 20 mM HEPES, pH 7.4 until used in electrophysiological recordings. Whole-cell patch clamp was used to record proton currents sealing at the cytoplasmic droplet from mature human spermatozoa plated on poly-lysine coated coverslips.
Pipettes (20-30 MΩ) were filled with 135 mM N-methyl-D-glucamine (NMDG), 5 mM ethylene glycol tetraacetic acid (EGTA), and 100 mM MES adjusted to pH 6.0 with methanesulfonic acid (CH3SO3H). Seals between the patch pipette and the sperm cytoplasmic droplet were formed in physiological solution. After transition into whole-cell mode, the bath solution was changed to one that was free of added divalent cations comprised of 130 mM NMDG, 1 mM EDTA and 100 mM HEPES, pH 7.4 with CH3SO3H to measure dose-dependent activation of proton currents by Alb (
After sperm sample liquefaction (30 minutes at 37° C.), highly motile sperm were recovered by swim-up separation for 1 hour in HTF (see above), at 37° C. in an atmosphere of 5% CO2-95% air. Non-capacitated sperm were diluted to 107 sperm/mL with modified HTF (mHTF: 4 mM NaHCO3 and 21 mM HEPES were used to replace 25 mM NaHCO3 in HTF) and loaded with BCECF (2 μM, cell-permeant, dual-excitation ratiometric fluorescent pH probe) in the dark at 37° C. for 30 min. Fluorescence intensity was measured with an Aminco Bowman II spectrofluorometer (λEx1=508, ×Ex2=450, ×Em=535). Non-capacitated sperm were allowed to stabilize until there was no change, or a small continuous change, in the fluorescent signal. Then Alb or Fab were added and changes in fluorescence were recorded for 400 s at a frequency of 0.5 Hz. High concentrations of Alb likely to cause quenching of fluorescent dyes, so 75 μM was the highest concentration tested. For the C6 group, non-capacitated sperm were incubated with 20 μM C6 for 1 h before the experiment. Conversion of the BCECF Ratio (508/450 nm) to pH value was performed using a calibration curve adjusted with a conventional pH electrode as previous described28. Statistical analyses were performed using the Dunnett Test.
Sperm Intracellular Ca2+ MeasurementsNon-capacitated sperm were diluted to 107 sperm/mL with mHTF and loaded with Fluo3-AM (2 μM, cell-permeant fluorescent Ca2+ probe) in the dark at 37° C. for 30 min. Non-capacitated sperm were allowed to stabilize before 75 μM Alb was added and the changes in fluorescence intensity were measured (λEx=505 and λEm=525) for 950 s at a frequency of 0.5 Hz. The increase in [Ca2+]i was triggered by adding 15 μM progesterone. Raw intensity values were normalized as [(F/F0)−1], where F is fluorescence intensity at time t and F0 is the mean of F taken during the control period. In some studies, 10 μM ionomycin was applied after progesterone to study the ionophore-mediated rise in [Ca2+]i and demonstrate that Alb did not alter dye loading; in this case normalization was to ionomycin induced peak [Ca2+]i. Statistical analyses were performed using the Dunnett Test.
Sperm Acrosome Reaction MeasurementsSperm suspensions were diluted to 107 sperm/mL with HTF and incubated 3-4 h at 37° C. in an atmosphere of 5% CO2-95% air in presence of 5 mg/mL BSA to promote capacitation, or in absence of BSA for non-capacitating conditions. The effect of different concentrations of Alb, in the presence or absence of 20 μM C6 were tested. Spermatozoa were incubated for 30 min at 37° C. with 15 μM progesterone, spotted on slides, air-dried and stained with FITC-coupled Pisum sativum agglutinin (FITC-PSA, 25 pg/mL in PBS) for 40 min at room temperature. The presence of an intact acrosome was assessed in at least 200 cells per condition using an upright Nikon Optiphot II microscope equipped with epifluorescence optics. Statistical analyses were performed using Dunnett Test.
Neutrophil ROS MeasurementHuman neutrophils were isolated from peripheral blood and re-suspended in Hank's Balanced Salt Solution (HBSS) comprising 138 mM NaCl, 5.4 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2), 0.5 mM MgCl2, 0.4 mM MgSO4, 4.2 mM NaHCO3, 5.5 mM glucose and 20 mM HEPES, pH 7.2, and dispensed into white 96-Well Immuno Plates (2×105 cells/well). Neutrophils were incubated with 500 μM Luminol (Sigma) and different concentrations of Alb for 30 min at 37° C. For the C6 group, neutrophils were pre-incubated with 20 μM C6 for 30 min before the incubation with Luminol and Alb. After incubation, neutrophils were stimulated with fMLF and the chemiluminescence was measured immediately every 1 min for 60 min using Fluoroskan FL (ThermoFisher).
Neutrophil Elastase MeasurementHuman neutrophils were washed and re-suspended in HBSS. Elastase release from neutrophils was evaluated using Elastase Substrate I, Colorimetric, AAPV-pNA (Millipore). All experiments were performed in polypropylene microcentrifuge tubes. Following the 30 min incubation with Alb or control protein Fab at 37° C. neutrophils (6.5×105 cells per tube) were subjected to stimulation using 1 μM fMLF. All cells were incubated for another 5 minutes and subsequently centrifuged at 400 g for 5 min. The cell free supernatant was added to individual microplate wells to achieve a total reaction volume of 200 μL per well and an AAPV-pNA concentration of 0.4 mM. Reactions were performed at 37° C. for 1 hour, following which absorbance was measured at 405 nm. An extinction coefficient of 8.8×103 cm/M was used to calculate the units of elastase released. This number was then divided by the total neutrophil elastase content as determined from the Triton X-100 incubated neutrophils yielding the percentage of total elastase release for each group.
Two Color smTIRF and Photobleaching
HEK293T cells were seeded on glass bottom dishes (Chemglass Life Science) and transfected with hHv1-G199L-TFP. The surface density of channel molecules was kept less than 200 in a 10×10 μm field to minimize the overlapping of multiple channels within a diffraction-limited spot. TAMRA-Alb was added in 100 mM HEPES, 90 mM NaCl, 10 mM KCl, 0.5 mM CaCl2), 1.2 mM MgCl2, and 10 mM glucose, pH 7.5 to the dishes and incubate 30 min for reaching binding equilibrium. Cells were extensively washed to remove nonspecifically bound TAMRA-Alb before recording. Single protein molecules or complexes at the surface of live HEK293T cells were identified using TIRF microscope as described5. The critical angle for TIRF was adjusted using a CellTIRF illuminator (Olympus) and a high numerical aperture apochromat objective (150×, 1.45 NA) mounted on an automated fluorescence microscope controlled by Metamorph software (Molecular Devices). Metamorph was used to simultaneously illuminate both fluorophores at a critical angle such that only 100 nm deep was illuminated. TAMRA was excited with the 561 nm laser line and TFP was excited with a 445 nm laser line. Emitted light signals were split with a 520 nm dichroic mirror mounted in a DualView adapter (Photometrics), which allows each wavelength to be directed to one half of a back-illuminated EM-CCD. Stoichiometry was assessed by simultaneous photobleaching with continual excitation. Data was captured as movies of 100-370 frames acquired at 1 Hz.
Data was analyzed as previously described5. When TAMRA was with TFP in the same cell, the data for each fluorophore were saved as separate stacks and processed in an identical manner. The Manders' coefficient of colocalization (MMC) between fluorophores was determined by unbiased intensity correlation analysis using the Coloc2 plugin in ImageJ to confirm overlap of the two molecules. Fluorescence measured from each region was plotted versus time to determine the number of bleaching steps at each point. Statistical analyses to calculate estimated confidence with which stoichiometry could be inferred from the observed data and θ, the probability of successfully observing each possible photobleaching event, were performed in R Studio, based on methods developed by Hines66. The densities of colocalized and single fluorescent spots were determined following thresholding and watershed separation in ImageJ. Then the particle number was counted in separate regions for 3-5 cells per group using the Analyze particles plugin.
Single-molecule photobleaching events are missed in practical application to biological systems because of fluorophore prebleaching, the quantum efficiency of the fluorophore, and the time resolution of smTIRF system; in our studies, the error is estimated to be less than 10% as previously demonstrated5, 46.
Live Cell FRET MicroscopyDonor-decay time-course was studied as before45, using an Olympus inverted epi-fluorescence microscope. HEK293T cells were seeded on glass bottom dishes (Chemglass Life Science) and transfected with hHv1-TFP and T-Alb-VFP variants. Cells were recorded in a solution comprising 100 mM HEPES, 90 mM NaCl, 10 mM KCl, 0.5 mM CaCl2), 1.2 mM MgCl2, and 10 mM glucose, pH 7.5. Resting membrane potential (RMP) was altered by isotonic replacement of extracellular NaCl with KCl. For EGTA studies, 1 mM was added to the 10 mM KCl bath solution. TFP was excited at 445 nm and the emission collected through a 470-500 nm bandpass filter, VFP was excited at 514 nm and the emission collected through a 525-575 nm filter. Images were captured using a scientific camera controlled by Metamorph software (Molecular Devices) and were analyzed with ImageJ as described, 45.
Protein-Peptide in Silico DockingUnguided docking was performed using the amino acid sequence of the S3-S4 loop peptide of hHv1 (F190 to L200) and the crystal structure of Alb (PDB: 1BMO), to predict potential binding sites, using HPEPDOCK web server48.
Molecular Dynamics SimulationsThe initial structure of the dimeric hHv1 (only the transmembrane region: residues G90 to 1218) at resting state was adopted from our previous study where the model was generated and refined by molecular dynamics (MD) simulations using spectroscopic constraints43. The hHv1 modeling structure was embedded in an explicit POPC lipid bilayer using the program VMD69, while the crystal structure of Alb (PDB 1BMO; residues S5 to A582) was placed in the extracellular side with its DII domain and the two potential binding sites facing the S3-S4 loops of hHv1, fully solvated in a 100 mM KCl solution. The final system contained approximately 185,000 atoms and was electrically neutral. The H193 on the subunit A of hHv1 was modeled in a protonated state to form hydrogen bonding interactions with E188 and E292 of Alb. Of note, the two glutamate residues could be the potential hydrogen bond donors as well.
The system was initially minimized for 5,000 steps, followed by 10 ns equilibration with positional restraints (0.5 kcal/mol/Å2) being applied to the backbone of the whole Alb and the transmembrane helices of hHv1 to relax with the lipids. Then, the positional restraints were removed except the intracellular half of the transmembrane helicesS4 of hHv1 (residues R205 to I218), which were used to maintain the dimeric interface of hHv1 as its cytosolic coiled-coil structure was excluded from the model to minimize the size of the system. The distance of center of mass between the side-chain heavy atoms of hHv1-H193 (subunit A) and Alb-H288, and hHv1-H193 (subunit B) and Alb-H338 was gradually decreased (approaching to 5 Å) in 10 ns to pull Alb toward hHv1 using the Colvars module70. Meanwhile, the S1-S2 loops of the dimeric hHv1 were given a distance restraint for optimized Alb contact with hHv1.
After that, 150 ns simulations were performed with different combinations of distance restraints between hHv1-H193 and their potential partners on Alb (Supplementary Table 4) to explore the interacting networks at the two binding sites and other contact regions of the complex where spontaneously and transiently formed hydrogen bonds between hHv1 and Alb had been found during the simulations. We used the distance restraints without angle restraints to determine if the enforced interaction was favorable. If the restrained interaction was favorable, a correct hydrogen-bonding angle formed automatically (Supplementary
During the simulations, we found that E294 and E297 on Alb were close to 1202 of hHv1, a helix turn upward the first gating charge R205, making E294 and E297 the probable negative countercharges activating the outward movement of the S4 helices of the dimeric hHv1. Thus, we generated and simulated another system of the Alb and hHv1 complex using the “up” state hHv1 (residues G90 to K221) from our previous study43. The simulation protocol was similar to the resting state model system with a total simulation time of 150 ns.
The MD simulations were carried out in the periodic boundary conditions with a time step of 2 fs using the NAMD software program49. The CHARMM36 parameter set71 was used for proteins, lipids and ions, and the TIP3P model for water. The temperature and pressure were constrained at 300 K and 1 atm, respectively, using the Langevin dynamics and the Nose-Hoover Langevin piston method71. The long-range electrostatic force was calculated with the particle-mesh Ewald method73, and the short-range electrostatic and van der Waals interactions were smoothly switched off at 10-12 Å.
The evaluate the stability of the binding interactions of the two proteins, the two systems were further subjected to microsecond scale long simulations with ANTON250, a special-purpose computer for long time scale MD simulations (Supplementary
Statistical analyses were performed using the Dunnett Test, *P<0.05, **P<0.01, ***P<0.001. Data are presented, where indicated as the mean±standard error of the mean (SEM). The number of replicates for each study are described in the legends.
Figures LegendsAs described in the Methods, mature human spermatozoa were collected and recorded by whole-cell patch clamp or studied using spectrofluorimetry. Alb in all studies was fatty acid-free. Values are mean±SEM, n=3-4 cells (patch clamp) or independent experiments for each condition. 107 sperm were used in each measurement. Statistical analyses were performed using the Dunnett Test, *P<0.05, **P<0.01, ***P<0.001.
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- a, Left, Representative proton current traces in non-capacitated human sperm in the absence (top) and presence (bottom) of 80 μM Alb in response to test pulses from −60 and +60 mV in 20 mV increments from a holding voltage of −60 mV every 10 s with a approximately 30-fold proton gradient (pHi=6.0 and pHo=7.4) using divalent cation-free bath solution. Right, hHv1 currents measured at the end of a test pulse to +60 mV after exposing sperm to 80 μM Fab (black), 80 μM Alb (grey), or 80 μM Alb+1 μM C6 (red). Values are normalized to mean proton current amplitude in the absence of Alb. The C6 peptide blocked 86±3% of the proton current activated by Alb.
- b, Dose-response relationships for Alb potentiation of sperm proton current at depolarization voltage of +60 mV. The half maximal effective concentration (EC50) of Alb for activating sperm proton current was estimated from the fit to Hill relationship as 158±16 μM with a Hill coefficient of 1.09±0.01. Values are normalized to mean proton currents amplitude in the absence of Alb.
- c, Conductance-voltage relationships (G-V) for sperm proton currents in the absence (▪) or presence of 800 μM Alb (●). The proton conductance in sperm showed a −32±3 mV shift after exposure to 800 μM Alb from 4±1 mV to −28±2 mV (Supplementary table 5).
Curves were fit to a Boltzmann equation, as described in Methods. d, Left, Non-capacitated sperm were loaded with the fluorescent ratiometric pH probe BCECF and changes of fluorescence intensity were measured. 75 μM Alb (grey) increased pHi and the increase was inhibited by 20 μM C6 (red). 75 μM Fab alone had no effect (black). Right, BCECF fluorescent signals were converted to ΔpH according to the lineal relation between fluorescence ratio values (RF508/F450) and pHi values as described (Methods). Alb-induced cytoplasmic alkalization in a concentration dependent manner (grey). The Alb-triggered pHi increase was inhibited by C6 (red) and Fab did not increase the pHi (black) (and Supplementary
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- e, Non-capacitated sperm were loaded with the fluorescent intracellular calcium indicator Fluo-3 and changes in fluorescence were measured. Alb potentiates the increase of [Ca2+]i induced by progesterone (15 μM) in a concentration dependent manner (grey bars). The [Ca2+]i increase potentiated by 75 μM Alb was 2.3-fold and this was fully suppressed by 20 μM C6 peptide (red bar) (and Supplementary
FIG. 1b, c ). - f, Acrosomal exocytosis induced by 15 μM progesterone was evaluated in diluted human sperm suspensions under different conditions as described (Methods). Upon progesterone stimulation, control capacitated sperm underwent the acrosome reaction (Cap, black bar), whereas non-capacitated sperm did not (Non-Cap, black bar). Incubation of C6 peptide (20 μM) with non-capacitated sperm had no effect (C6+Non-Cap, black bar). In a concentration dependent manner, incubation with Alb increased the progesterone-induced acrosome reaction (grey bars) in non-capacitated sperm; addition of C6 (20 μM, red bar) fully inhibited exocytosis stimulated by 75 μM Alb.
- e, Non-capacitated sperm were loaded with the fluorescent intracellular calcium indicator Fluo-3 and changes in fluorescence were measured. Alb potentiates the increase of [Ca2+]i induced by progesterone (15 μM) in a concentration dependent manner (grey bars). The [Ca2+]i increase potentiated by 75 μM Alb was 2.3-fold and this was fully suppressed by 20 μM C6 peptide (red bar) (and Supplementary
Human neutrophils were isolated from peripheral blood of healthy volunteers and recorded by whole-cell patch clamp or studied using microplate luminometer (Methods). Values are mean±SEM, n=3-4 human neutrophils (patch clamp) or independent experiments with neutrophils at the numbers indicated.
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- a, Representative proton currents via hHv1 in human neutrophils studied in the absence (left) and presence (middle) of 450 μM Alb in response to test pulses from −60 and +60 mV in 20 mV increments, from a holding voltage of −60 mV every 15 s with a approximately 30-fold proton gradient (pHi=6.0 and pHo=7.5) in divalent cation-free bath solution. Fitting the activation and deactivation of proton currents at +60 mV to a single exponential gave time constants that are reported in Supplementary table 1. Right, hHv1 currents measured at the peak of a test pulse to +60 mV after exposing neutrophils to 450 μM Fab (black), 450 μM Alb (grey), or 450 μM Alb+20 μM C6 (red). Values are normalized to mean proton current amplitude in the absence of Alb. The C6 peptide blocked 87±2% of of the proton current activated by Alb.
- b, Left, effect of fMLF alone or in combination with Alb on ROS production by human neutrophils. Human neutrophils (2×105 cells) were incubated in HBSS without or with 450 μM Alb for 30 min at 37° C., then stimulated with 1 μM fMLF. ROS was measured using luminol, a chemiluminescent substrate. ROS production from unstimulated neutrophils is negligible (o). Alb (●) enhanced and sustained the fMLF-stimulated ROS production compared to neutrophils stimulated with fMLF alone (●). Incubation with 20 μM C6 (●) inhibited ROS production potentiated by 450 μM Alb. Right, ROS produced 60 min after fMLF stimulation. In a concentration dependent manner, incubation with Alb increased the fMLF-stimulated ROS production by human neutrophils (grey bars); addition of C6 (20 μM, red bar) inhibited the ROS production potentiated by 450 μM Alb. 450 μM Fab had no effect on fMLF-stimulated ROS production (black bar). Values are normalized to mean ROS production by neutrophils stimulated with fMLF alone.
- c, Human neutrophils (2×105 cells) were incubated without (0) or with 450 μM Alb (0) and then stimulated with increasing concentrations of fMLF. ROS were measured as described in panel b. The potentiation by Alb of total ROS production became appreciable at 10 nM fMLF and increased with increasing fMLF (Supplementary
FIG. 3d ). Some error bars are smaller than symbols. - d, Human neutrophils (6×105 cells) were incubated with Alb for 30 min at 37° C. and then stimulated with 1 μM fMLF. Total elastase release at each test condition was measured spectrophotometrically (Methods). The values are normalized to elastase release stimulated with fMLF alone (Control, black). 450 μM Alb increased the elastase release by neutrophils (grey), and the increase was inhibited by 20 μM C6 (red) whereas 450 μM Fab had no effect on fMLF-stimulated elastase release (black).
hHv1 channels were expressed in HEK293T cells and studied by whole-cell patch clamp to determine biophysical parameters using 1.5 s test steps to 0 mV from a holding voltage of −60 mV every 10 s with a 10-fold proton gradient (pHi=6.5 and pHo=7.5) and divalent cation-free bath solution, as described in Methods. Values are mean±SEM, n=6-8 cells for each condition. Some error bars are smaller than symbols.
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- a, Representative proton current traces for hHv1 channels before (left), and in the presence of 75 μM Alb (right), with steps of 20 mV increments from −60 mV to +40 mV. The current measured at the end of depolarization was used to determine the extent of activation. Fitting the activation and deactivation of proton currents at 0 mV to a single exponential function gave time constants τ−act of 2619.8±222.3 ms and 332.3±30.6 ms, τ−tail of 86.9±8.4 ms and 235.0±29.7 ms without and with Alb, respectively.
- b, G-V for hHv1 in the absence (▪) or presence of 75 μM Alb (●). hHv1 channels showed a −23±3 mV shift after exposure to 75 μM Alb from 15±2 mV to −8±1 mV. Curves are fitting to the Boltzmann equation as described in Methods.
- c, Time course for activation and deactivation of hHv1 on acute application (bar) and washout of 75 μM Alb. Currents were recorded at 0 mV. Values are normalized to the control current before the application of Alb.
- d, Dose-response relationships for Alb activation of hHv1 at depolarization voltage of 0 mV. The EC50 of Alb for hHv1 channels was estimated from the fit to Hill relationship as 74.8±8.7 μM with a Hill coefficient of 1.16±0.11. Values are normalized to mean proton currents amplitude in the absence of Alb.
- e, Cartoon showing two C6 peptides binding to an intact dimeric hHv1 channel.
- f, 20 μM C6 peptide (red trace) was applied to cells after control pulses (black trace) without Alb (left) or after pre-activation with 75 μM Alb (right). C6 inhibited approximately 92% of proton current through hHv1 (left), which is similar to the extent of inhibition (approximately 89%) with Alb pre-activation (right).
hHv1, CiHv1, hHv1-G199L and chimeric channels were expressed in HEK293T cells and studied by whole-cell patch clamp as described in
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- a, Top, sequence alignments of the S1-S2 and S3-S4 motifs of hHv1 (black), CiHv1 (blue) and loop-transplant chimeras. The S1-S2 or/and S3-S4 loop of hHv1 or CiHv1 (I121 to F139 and I183 to L204, respectively, numbered according to hHv1) were transplanted into the corresponding region of CiHv1 or hHv1 to produce functional chimeric channels, as described (Methods). Bottom, representative proton current traces at 0 mV for channels in the absence (black trace) or presence of 75 μM Alb (red trace).
- b, Activation by Alb (75 μM) of WT and chimeric channels, current at the end of a test pulse to 0 mV is plotted. Values are normalized to the control current amplitude before the Alb application. The cartoons represent the topology of a single subunit with hHv1 segments in black and CiHv1 segments in blue). The S3-S4 loop of hHv1 confers Alb sensitivity to hS3-S4CiHv1 channel chimeras.
- c, A mutagenesis scan of the S3-S4 loop of hHv1 (substitution with Cys except for V187A, L189A and L203A) showing changes in Alb activation; the numbering corresponds to the amino acid sequence of hHv1. Each mutant was examined with Alb at 75 μM and measured with tests pulse to 0 mV. Values are normalized to the control currents amplitude before the application of Alb.
- d, Left, representative proton current traces for hHv1-G199L channels in the absence (up) and presence of 1 μM Alb (bottom) with steps of 20 mV from −60 mV to +40 mV. Right, Alb dose-response relationship for hHv1-G199L provides an EC50 for Alb activation of 3.4±0.5 μM when estimated by the fit to a Hill relationship with a coefficient of 0.93±0.10. Values are normalized to mean proton current amplitude in the absence of Alb. Some error bars are smaller than symbols.
- e, Cartoon of one TAMRA-Alb molecule binding to the two subunits in a hHv1-G199L-TFP channel.
- f, Left, incubation of TAMRA-Alb (red) with heterologously expressed hHv1-G199L-TFP (teal) results in single colocalized particles (white) with both TAMRA and TFP fluorescence at the surface of cells (yellow circles). Middle, montage of photobleaching time course of a single fluorescent particle (indicated by arrow in left panel) during continuous excitation to bleach the fluorophores (every fifth frame shown). Right, time courses for simultaneous photobleaching of both fluorophores in the particle, showing one stepwise change in fluorescence intensity for TAMRA-Alb and two for hHv1-G199L-TFP (arrows).
- g, Histogram of photobleaching steps for hHv1-G199L-TFP (teal bars) and TAMRA-Alb (red bars) on simultaneous photobleaching. 89% of studied particles with hHv1-TFP were bleached in 2 steps. The data analyzed by the approach of Hines66 estimate hHv1-G199L channels in surface particles formed with two subunits, with an assessed confidence of >0.999 (Supplementary Table 2). Among all colocalized particles containing both fluorescent colors, 95% have one TAMRA-Alb bleaching step at 3 μM TAMRA-Alb (Supplementary Table 3).
Tethered Alb and other tethered constructs carried VFP were designed as described (Methods). The VFP constructs were co-expressed with hHv1-TFP or CiHv1-TFP in HEK293T cells and studied by FRET microscopy. FRET between intracellular VFP and TFP resulted from the extracellular interaction of Alb (and variants) or C6 with hHv1 and was assessed by measuring the mean time constant (τ) for TFP (donor) photobleaching in the presence of VFP (acceptor) from 3 regions of 6 cells per group. Values are mean±SEM.
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- a, Schematic representation of T-Alb-VFP showing binding of Alb to the hHv1-TFP channel. T-Alb-VFP is a fusion protein of Alb with an N-terminal signal peptide (naturally cleaved after expression), Alb protein (orange), a hydrophilic flexible linker, the transmembrane domain of the platelet-derived growth factor receptor (yellow), and the monomeric yellow fluorescent protein, mVenus at the intracellular C-terminus. Extracellular interaction of Alb and hHv1 brought the TFP and VFP fluorophores into close proximity (<10 nm), resulting in FRET that is quantitated by changes in donor photobleaching.
- b, Representative current traces (studied as in
FIG. 2a with steps from −60 to +40 mV) for hHv1-TFP channels without (Control) or with co-expression of T-Alb-VFP, T-NO-VFP (no Alb inserted) and T-C6-VFP. When co-expressed with hHv1-TFP, T-Alb-VFP activates while T-C6-VFP fully inhibits the outward proton current. Co-expression with T-NO-VFP had no effect comparing with hHv1 alone. - c, Whole-cell peak current density of hHv1-TFP channels measured at 0 mV without (Control) or with co-expression of T-NO-VFP, T-Alb-VFP and T-C6-VFP are plotted from the experiments shown in panel b.
- d, G-V relationship for hHv1-TFP without (▪) or with co-expression of T-Alb-VFP (●). Curves are fit to the Boltzmann equation as described (Methods).
- e, hHv1-TFP (cyan) and T-Alb-VFP (yellow) are shown to reach the cell surface. Scale bar is 10 m. The boxed area of membrane shows donor photobleaching with continuous illumination.
- f, Exemplar photobleaching showing the decay of fluorescence intensity for regions of single cells expressing hHv1-TFP (▪) or hHv1-TFP with T-Alb-VFP (●). τ was determined by single-exponential fits to the time course of photobleaching.
- g, FRET showed that hHv1-TFP (black bars) assembled with T-Alb-VFP and T-C6-VFP but not with T-NO-VFP. In contrast, CiHv1-TFP (white bars), a proton channel that is not activated by Alb or inhibited by C65, did not show FRET with T-Alb-VFP and T-C6-VFP.
- h, Tethered Alb domain variants (T-DI-VFP, T-DII-VFP, T-DIII-VFP, T-DI-DII-VFP, T-DII-DIII-VFP and T-DI-DIII-VFP) were produced as described in Methods and co-expressed with hHv1-TFP for FRET studies. τ of photobleaching for each pair was normalized to the ≢ of photobleaching of hHv1-TFP alone (Control).
- i, T-Alb-VFP with mutations were designed based on two predicted Alb sites as hot spots for interaction with S3-S4 loop peptide of hHv1 from in silico docking analysis (Supplementary
FIG. 13 ), produced as described in Methods, and co-expressed with hHv1-TFP for FRET studies. τ of photobleaching for each pair was normalized to the T of photobleaching of hHv1-TFP alone (Control).
The structural model of Alb-hHv1 complex was generated using the VMD software program, using the crystal structure of Alb (PDB 1BMO) and our reported modeling structure of dimeric hHv1 at rest43. The final complex model was equilibrated and refined by MD simulations in a hydrated lipid bilayer, using residue-residue interactions suggested by in silico docking (Supplementary
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- a, The Alb-hHv1 complex in two orientations showing the three Alb domains DI (grey), DII (orange) and DIII (wheat) and the two subunits (cyan and blue) of a dimeric hHv1 channel. The horizontal gray lines indicate the position of the membrane boundary.
- b, Close-up view of the interaction interface between Alb and two individual hHv1 subunits in two binding sites in the structural model. Left, interaction Site 1, H193 of hHv1 subunit A (cyan) may form direct interaction with Alb residue H288 and the adjacent E188 and E192. Right, interaction Site 2, H193 of hHv1 subunit B (blue) may form direct interaction with Alb residue H338 and the adjacent Y334, R337 and F374.
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- a, The higher level of Alb in the female reproductive tract activates hHv1 in sperm leading to H+ efflux and cytosolic alkalization, initiating capacitation by potentiating CatSper. The synergy of hHv1 and CatSper in human sperm capacitation has been described5-7.
- b, Activation of hHv1 in neutrophils by Alb potentiates H+ efflux, maintaining physiological pHi and compensating for the depolarization caused by NOX2, enhancing enzyme activity and allowing sustained ROS production. During the neutrophil respiratory burst (here, stimulated by fMLF), NOX2 translocates electrons from NADPH across the membrane to reduce O2 to O2−; this efflux of electrons generates H+ in the cytoplasm and depolarizes cells, inhibiting the activity of NOX25, 8-10.
- c, Alb activation of hHv1 is proposed to proceed by an electrostatic gating modification mechanism. Two binding sites in the Alb DII domain interact with the two S3-S4 loops in hHv1 channels. The two Alb sites each have an adjacent negatively-charged residue (Alb-E294 and Alb-E297) that attracts the first channel gating charge (hHv1-R205) to facilitate outward movement of the voltage sensors and stabilizes the sensors in the activated “up” conformation. Side chains of three gating charges (R205, R208 and R211), the “hydrophobic gasket” formed by F150 that separates the internal and external aqueous solutions67, and 1202, one helical turn above R205, are shown on hHv1 subunit A (cyan).
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Supplementary table 1. Parameters for Alb activation of hHv1 in HEK293T cells, human sperm and neutrophils, illustrated in
Supplementary table 2. Stoichiometry of hHv1-G199L-TFP channels: hHv1-G199L-TFP was expressed in HEK293T cells and studied by smTIRF (Methods). The number of photobleaching steps observed for TFP in each single fluorescent spot reports on the stoichiometry of channels. Like WT hHv15, hHv1-G199L-TFP channels are dimeric. Analysis was performed according to methods described by Hines66. The statistical confidence in the null hypotheses that hHv1-G199L-TFP forms dimers was assessed to be greater than 0.999. Prebleaching and variance in quantum efficiency reduce the probability of observing each possible bleaching event (0). 0 is calculated from the value of n and the distribution of the photobleaching data66. θ is decreased when the distribution is altered to estimate the possibility that higher numbers of missed bleaching steps, for example θ+1, indicating that this stoichiometry is less likely.
Supplementary table 3. Parameters of single molecule photobleaching with hHv1-G199L TFP and TAMRA-Alb by smTIRF: hHv1-G199L-TFP was expressed in HEK293T cells and incubated with 3 μM TAMRA-Alb to reach equilibrium. Simultaneous, two-color, single particle photobleaching was studied by TTRF (Methods). Mean Manders' coefficient of colocalization were generated by using unbiased intensity correlation analysis as described in the Methods. The number of colocalized particles and photobleaching steps observed for TFP and TAMRA fluorophores in each single fluorescent spot were analyzed as previously described5 and used for determining the binding stoichiometry of Alb with channels.
Supplementary table 4. Distance restraints between pairs of residues used in the molecular dynamics simulations: The distance between two selected atoms or centers of mass of two groups of atoms was harmonically restrained with a force constant of 5 kcal/mol/Å2 or less, centered at 3.5 Å for the hydrogen bond donor/accepter pairs and 4 or 5 Å for the rest. Different combinations of the restraints were applied during the refinement process. The restraints applied in the ANTON2 simulations were highlighted in red.
Supplementary table 5. Impact of EDTA and EGTA on the voltage-dependence of hHv1 activation (V1/2) with and without Alb in sperm and HEK293T cells: Proton currents in human sperm were studied by whole-cell patch clamp, as described in
Mature human spermatozoa were collected and studied using spectrofluorometry. pHi and [Ca2+]i were determined in non-capacitated sperm as described in Methods using BCECF and Flour-3, respectively. Values are the mean±SEM for 3-5 independent experiments. Reagent included 15 μM progesterone (Pg), 75 μM Alb or bovine serum albumin (BSA), the Fab fragment of human IgG (Fab) at 20 μM, C6 at 20 μM, the CatSper channel blocker NNC at 1 μM, the Ca2+ ionophore ionomycin (iono) at 10 M, Statistical analyses were performed using the Dunnett Test, **P<0.01, ***P<0.001.
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- a, pHi changes were measured with BCECF and converted to ΔpH as described in Methods. Alb increased the speed of cytoplasmic alkalization in a concentration dependent manner. C6 inhibited intracellular alkalization induced by Alb (red) whereas did not (black).
- b, The increase in [Ca2+]i triggered by Pg was measured in the absence of Alb (left) and after adding Alb (middle). The fluorescence increase stimulated by Pg in presence of Alb was inhibited by C6 (right).
- c, The presence of Alb (grey bar) increased peak [Ca2+]i stimulated by Pg 2.4-fold over baseline and this increase was inhibited by C6 (red bar) or NNC (blue bar).
- d, Representative recordings of [Ca2+]i without (left) or with (right) BSA in arbitrary fluorescence units (au). BSA enhanced [Ca2+]i influx induced by Pg. The absolute rise in au on subsequent addition of iono was similar with BSA (0.31±0.05 au) and without BSA (0.39±0.03 au) indicating similar dye loading in the two conditions.
- e, [Ca2+]i changes normalized to peak au on addition of iono.
- f, Five independent samples of human sperm with BSA (right, grey) showed a 2.2-fold increase in peak [Ca2+]i compared to control (left, black) here calculated as ΔPg/Δiono, where ΔPg and Δiono are the change in peak [Ca2+]i induced by Pg and iono, respectively.
- g, pHi changes were measured as described in panel a. BSA increased the speed of cytoplasmic alkalization and BSA pre-saturated and applied with a 10-fold molar excess cholesterol had a similar effect on the rate of pHi increase as did untreated BSA alone.
A typical voltage-ramp protocol was used for recording monovalent CatSper current in whole-cell voltage-clamp experiments with non-capacitated human sperm, as described in Methods.
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- a, The representative monovalent CatSper current trace in the absence (black trace) and presence (red trace) of 80 μM Alb, or in presence of 10 mM NH4Cl (blue trace) are shown.
- b, Relative current amplitudes measured at +80 and −80 mV from experiments as shown in panel a. Values are mean±SEM, n=4.
As described in Methods, human neutrophils were isolated from peripheral blood of healthy volunteers and recorded by whole-cell patch clamp or measured using a microplate luminometer for ROS production. Values are mean±SEM, n=3 cells or independent experiments for each condition.
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- a, Proton currents via hHv1 in human neutrophils were studied as described in
FIG. 2a with a approximately 30-fold proton gradient (pHi=6.0 and pHo=7.5). G-V for neutrophils proton currents in the absence (▪) or presence of 450 μM Alb (●). The proton conductance in neutrophils showed a −35±3 mV shift after exposure to 450 μM Alb. Curves are fitting to the Boltzmann equation as described in Methods. - b, The ROS production was measured as described in
FIG. 2b . fMLF alone (1 μM) stimulated ROS production by human neutrophils which could be inhibited by 20 μM C6, while 450 μM Alb alone did not increase ROS production comparing to unstimulated human neutrophils (Control). - c, Normalized ROS production by human neutrophils without (●) or with the incubation of 450 μM Alb (●) stimulated with an increasing concentration of fMLF equivalent to the
FIG. 2c . Values of the total ROS production were normalized such that the ROS production stimulated by 0.01 nM and 10 μM fMLF correspond to 0 and 1, respectively. Some error bars are smaller than symbols. - d, Effect of 10 μM fMLF alone or in combination with 450 μM Alb on ROS production by human neutrophils. Incubation with Alb enhanced and sustained the ROS production by human neutrophils
- a, Proton currents via hHv1 in human neutrophils were studied as described in
hHv1-T29A channels, carrying change in PKC-6 phosphorylation site at the N terminus of hHv1 were expressed in HEK293T cells and studied using whole-cell voltage clamp with 1.5 s pulses from a holding voltage of −60 mV with 10 s interval and a 10-fold proton gradient (pHi=6.5 and pHo=7.5). 75 μM Alb (red trace) was applied after control pulses (black trace). Alb-activation increases hHv1-T29A currents of approximately 3-fold at 0 mV (n=3).
hHv1 channels expressed in HEK293T cells were studied using whole-cell voltage clamp with 1.5 s pulses from a holding voltage of −60 mV with 10 s interval and a 10-fold proton gradient (pHi=6.5 and pHo=7.5). Values are mean±SEM, n=6-8 cells for each condition. Some error bars are smaller than symbols.
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- a, Representative proton current traces for hHv1 channels before (top), and in the presence of 500 μM Alb (bottom) with steps of 20 mV increments from −60 mV to +60 mV
- b, Conductance-voltage relationships (G-V) for hHv1 in the absence (▪) or presence of 500 μM Alb (●). hHv1 channels showed a 45±4 mV shift after exposure to 500 μM Alb (Supplementary table 1). Curves are fitted to the Boltzmann equation as described in Methods.
- c, Dose-response relationships for Alb on increasing of the activation time constants (T act) of hHv1 at depolarizing voltages of 0 mV and +60 mV. The activation of proton currents at 0 mV and +60 mV were fitted with a single exponential function to determine the extent of Alb activation on τact. Values of the ratio of τact in the presence and absence of Alb were normalized such that the ratio of τact in the presence of 1 μM Alb and 800 μM Alb values correspond to 0 and 1, respectively. The equilibrium affinity (Kd) of Alb for hHv1 channels activation at 0 mV and +60 mV was estimated from the fit to Hill relationship as 84.5±9.8 μM with a Hill coefficient of 1.08±0.13, and 54.6±6.7 μM with a Hill coefficient of 1.19±0.14, respectively.
hHv1 channels expressed in HEK293T cells were studied using whole-cell voltage clamp with 1.5 s pulses to 0 mV from a holding voltage of −60 mV with 10 s interval and a 10-fold proton gradient (pHi=6.5 and pHo=7.5).
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- a, 800 μM Fab (red trace) was applied after control pulses (black trace) and shows no activation of the proton current.
- b, Alb was incubated and digested with proteinase K at a concentration of 800 PM, as described in Methods. Alb after digestion (red trace) was applied after control pulses (black trace) and shows no activation of the proton current.
Alb does not activate other voltage-gated channels that were studied
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- a-d, Voltage gated potassium channels were expressed in HEK293T cells, and studied using 300 ms (hKv1.3, hKv2.1, hKv1.5) or 1.5 s (hIks) pulses to 0 mV from a holding voltage of −80 mV with 10 s interval. Bath solution comprises 130 mM NaCl, 4 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2), and 10 mM HEPES; pH was adjusted to 7.4 with NaOH. Electrodes were filled with a solution containing 130 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM K2ATP, and 10 mM HEPES; pH was adjusted to 7.4 with KOH. 75 μM Alb (red trace) was applied after control pulses and shows no activation comparing to control current before application (black trace).
- e, hNav1.5 was expressed in HEK293T cells, and studied using 20 ms pulses to 0 mV from a holding voltage of −100 mV with 10 s interval. Bath solution comprises 130 mM NaCl, 4 mM CsCl, 2 mM CaCl2), 1.2 mM MgCl2, 5.5 mM glucose, and 10 mM HEPES, pH 7.4 with NaOH. Electrodes were filled with a solution containing 60 mM CsCl, 80 mM CsF, 1 mM CaCl2), 1 mM MgCl2, 5 mM Na2ATP, 10 mM EGTA and 10 mM HEPES, pH 7.4 with CsOH. 75 μM Alb (red trace) was applied after control pulses and shows no activation comparing to control current before application (black trace).
hHv1 channels were expressed in Xenopus oocytes and studied by two-electrode voltage clamp (TEVC) with a holding voltage of −60 mV and 1.5 s steps to 100 mV every 10 s in the absence of proton gradient (pHi=pHo=7.3) as described in Methods. 75 μM Alb (red trace) was applied after control pulses (black trace) and increased hHv1 currents approximately 1.8-fold (n=3).
Hv1Sper was constructed by truncating 67 residues at the N-terminus of hHv1 (residues from A2 to R68). Hv1Sper channels expressed in HEK293T cells were studied using whole-cell voltage clamp with 1.5 s pulses from a holding voltage of −80 mV with 10 s interval and a 10-fold proton gradient (pHi=6.5 and pHo=7.5).
Values are mean±SEM, n=3 cells for each condition. Some error bars are smaller than symbols. Curves are fitted to the Boltzmann equation.
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- a, Representative proton current traces for Hv1Sper channels before (left), and in the presence of 500 μM Alb (right) with steps of 20 mV increments from −80 mV to +60 mV.
- b, G-V for Hv1Sper in the absence (▪) or presence of 500 μM Alb (●). Hv1Sper channels showed a 15±2 mV shift after exposure to 500 μM Alb (Supplementary table 1).
hHv1 channels expressed in HEK293T cells were studied using whole-cell voltage clamp with 1.5 s pulses from a holding voltage of −60 mV with 10 s interval and a 3-fold proton gradient (pHi=6.7 and pHo=7.2) in human tubal fluid (HTF) medium. Values are mean±SEM, n=3 cells for each condition. Some error bars are smaller than symbols.
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- a, Representative proton current traces for hHv1 channels in HTF medium before (left), and in the presence of 500 μM Alb (right), with steps of 20 mV increments from −60 mV to +60 mV.
- b, G-V for hHv1 in HTF medium in the absence (▪) or presence of 500 μM Alb (●). hHv1 channels showed a 40±4 mV shift in V1/2 after exposure to 500 μM Alb (Supplementary table 1). The voltage at which hHv1 channels start to open (Vthreshold) shifted from approximately 0 mV to −30 mV with Alb application. Curves are fit to a Boltzmann equation.
The hS3-S4Kv2.1 chimera channel was constructed as described in Methods. hKv2.1 and hS3-S4Kv2.1 were expressed in Xenopus oocytes and studied by TEVC with a holding voltage of −80 mV and 0.5 s steps to −20 mV every 10 s.
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- a, Sequence alignment of hKv2.1 (cyan), hHv1 (black) and hS3-S4Kv2.1 chimera transplanting the S3-S4 loop of hHv1 into hKv2.1.
- b, Representative current trace for hKv2.1 and hS3-S4Kv2.1 without (black) or with 500 μM Alb application (red). hKv2.1 was insensitive to 500 μM Alb, while the same concentration of Alb increased K+ current through hS3-S4Kv2.1 approximately 1.9-fold (n=3).
ΔHv1 monomeric channels appear to be insensitive, or weakly activated, by Alb
ΔHv1 channels were expressed in HEK293T cells and studied by whole-cell patch clamp to assess activation parameters using a holding voltage of −60 mV, 1.5 s test pulses, and a 10 s interpulse interval, pHi=6.5, pHo=7.5. Values are mean±SEM, n=3-8 cells for each condition. Some error bars are smaller than symbols.
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- a, Representative proton current traces for ΔHv1 channels before (left), and in the presence of 800 μM Alb (right) with steps of 20 mV from −60 mV to 40 mV. Fitting the activation and deactivation of proton currents at 0 mV to a single exponential function gave time constants Tact of 350±42 ms and 152±19 ms, T tan of 33±5 ms and 60±9 ms without and with 800 μM Alb, respectively. The observed activation is suspected to be due to a subpopulation of dimeric channels that forms with the truncated subunits.
- b, Dose-response relationships for Alb activation of ΔHv1. EC50 of Alb for ΔHv1 channels was estimated from the fit to Hill relationship as 1,904±155 μM with a Hill coefficient of 1.05±0.06. The maximal Alb (800 μM) potentiated currents amplitude on WT hHv1 (
FIG. 3d ) was set as the maximal effect for fitting
A computational protein-peptide docking web server (HPEPDOCK)48 was used to predict the binding configurations of a minimal 11-residue binding epitope (F190 to L200) within the S3-S4 loop of hHv1 on the Alb crystal structure (PDB: 1BMO). HPEPDOCK™ performed the protein-peptide docking through a hierarchical algorithm. Top 20 low energy models were favorably clustered in two binding sites, with 25% S3-S4 loop peptides clustered in Site 1, while 70% in Site 2 (three binding poses in Site 1 and two binding poses in Site 2 are shown). The distance between two predicated binding sites is approximately 27 Å, matching the distance (approximately 26 Å) between two individual hHv1 subunits of one dimeric channel.
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- a, Docking result for two low energy poses of S3-S4 loop peptide (cyan and lime) with Alb, showing binding Site 1 on DI (grey) and DII (orange) of Alb is one hot spot for hHv1 channel interaction.
- b, Close-up view of interaction interface between S3-S4 loop peptide and Alb in Site 1 in a docking model. The hHv1-H193 (cyan) could be a partner to Alb-E188, Alb-H288 and Alb-E292.
- c, Docking result for three low energy poses of S3-S4 loop peptide (blue, green, and red) with Alb, showing binding Site 2 on DII (orange) of Alb is the other hot spot for hHv1 channel interaction.
- d, Close-up view of interaction interface between S3-S4 loop peptide and Alb in Site 2 in a docking model. The hHv1-H193 (blue) could be a partner to Alb-Y334, Alb-R337, Alb-H338, Alb-D340, Alb-F374 and Alb-V381.
Starting configuration of Alb and hHv1 for all-atom MD simulations. The Alb, hHv1 and water were shown in a surface representation, and the lipid molecules and ions in spheres, respectively. The Alb was orientated with its DII (orange) facing the extracellular side of the dimeric hHv1 (cyan for subunit A, blue for subunit B). Some lipid molecules were removed for the sake of clarity.
Restraints were persistently used during long-term stimulation in order to maintain the Alb-hHv1 complex (Methods). The reasons were (1) the large thermal fluctuation of Alb. The size of Alb is very large, comprising of 578 residues in the model, compared with its binding partner the S3-S4 loop of hHv1 which is about 10 amino acids long. (2) Only the H193 residue on hHv1 was actively involved in the residue-residue interaction networks with Alb at channel “down” state. (3) The steric clash between the third domain of Alb and the S1-S2 loop of hHv1. The root-mean-square deviation (RMSD) analysis and principle component analysis (PCA) of the 1.5 s ANTON2 trajectories showed that there is a dramatic steric clash between Alb-DIII and the S1-S2 loop of hHv1 (Supplementary
Due to the large size of the system (approximately 185,000 atoms), we cannot simulate the complete binding/unbinding process to further evaluate the binding conformation. However, our RMSD and PCA results based on the multi-microseconds long restrained simulations showed that the global binding conformation between Alb and hHv1 is stable considering the big size of Alb, the flexibility of the S3-S4 loop of hHv1 at the binding interface, and only a few distance/positional restraints surrounding hHv1-H193 have being applied. In addition, by analyzing the last 0.5 s ANTON2™ trajectories, we found that the important hydrogen binding and π-π stacking interactions shown in
Time series of the backbone RMSDs of Alb and its three domains Alb-DI, Alb-DII, Alb-DIII in reference to the corresponding initial (red), final (blue) and crystal (black) structures in the 1.5 s ANTON2 simulation of the Alb-hHv1down system (a) and the Alb-hHv1up system (b). No initial structural alignment was performed in the RMSD calculations using the initial and final structures as reference to show the movement of Alb during the MD simulations. The vertical dashed line highlights the time boundary of two periods of the simulation. Top-view (left panel) and side-view (right panel) of the final structure of the Alb-hHv1down complex (c) and the Alb-hHv1up complex (d). The crystal structure of Alb was superimposed in transparent ribbon representation, showing the distortion of the Alb-DIII domain due to its steric clash with the S1-S2 loop of hHV1 (highlighted in the dashed box). The subunit A of hHv1 and the Alb-DI domain are not shown in the side-view for clarity
Principal component analysis (PCA) was performed on Cartesian coordinates of the Ca atoms of Alb generated in the 1.5 μs ANTON2 simulation of the Alb-hHv1down system (a, b, c) and the Alb-hHv1up system (d, e, f). Structural alignment based on the backbone atoms of the DII domain of the crystal structure was performed before the PCA analysis of the intra-molecular conformational changes of Alb (b, c, e, f), while no initial structural alignment was performed for the PCA analysis of the global conformational changes of Alb (a, d). The projections of all the conformations (n=2084) onto the first two principal components, PC1 and PC2, are shown as heat maps (a, b, d, e). Projections of the initial, final and crystal structures onto PC1 and PC2 are depicted as scattered points. c, f, Comparison of the square displacement of each residue along PC1 and PC2.
Snapshots from the last 500 ns ANTON2 trajectories of the Alb-hHv1down (a) and Alb-hHv1up (b) systems were used for the calculation (n=2084). The number in the parentheses represents the hydrogen bond propensity between the two residues. A hydrogen bond is defined as the distance between the hydrogen donor and acceptor atoms is shorter than 3.5 Å and the angle between the donor, the hydrogen and the acceptor atoms is larger than 120°.
Snapshots from the last 500 ns ANTON2 trajectories of the Alb-hHv1down (a) and Alb-hHv1up (b) systems were used for the calculation (n=2084). The number in the parentheses represents the hydrogen bond propensity between the two residues. A hydrogen bond is defined as the distance between the hydrogen donor and acceptor atoms is shorter than 3.5 Å and the angle between the donor, the hydrogen and the acceptor atoms is larger than 120°.
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- a, hHv1 channels were expressed in HEK293T cells, and studied using whole-cell voltage clamp with 1.5 s pulses to 0 mV from a holding voltage of −60 mV with 10 s intervals with a 10-fold proton gradient (pHi=6.5 and pHo=7.5) and divalent cation-free bath solution. 75 μM Alb that was presaturated with sodium cholesteryl sulfate (red trace) was applied after control pulses (black trace) and increased hHv1 currents approximately 8-fold (n=3).
- b-c, hHv1-H193A and hHv1-H140A channels, carrying changes in either the Zn2+ binding histidine residue in the S3-S4 loop or in the S1-S2 loop, respectively, were expressed in HEK293T cells, and studied using whole-cell voltage clamp with 1.5 s pulses to 0 mV from a holding voltage of −60 mV with 10 s interval. 75 μM Alb (red trace) was applied after control pulses (black trace). The H193A mutation disrupts activation so 75 μM Alb has no effect on hHv1-H193A (panel b), same as shown in
FIG. 4c for hHv1-H193C; in contrast, mutation of the Zn2+ binding residue in the S1-S2 loop did not alter Alb-activation showing an increase in hHv1-H140A currents of approximately 6-fold (panel c) (n=3).
As described in the Methods, mature human spermatozoa were collected and recorded by whole-cell patch clamp. G-V for sperm proton conductance showed a −53±5 mV shift in V1/2 after exposure to 500 μM Alb in EDTA free recording solution from 15 2 mV to −38±3 mV (Supplementary table 2). The Vthreshold shifted from approximately-30 mV to −70 mV with Alb application (Supplementary table 1). Curves are fitting to the Boltzmann equation as described in Methods. Values are mean±SEM, n=5 cells
hHv1 channels expressed in HEK293T cells were studied using whole-cell voltage clamp with 1.5 s pulses from a holding voltage of −60 mV with 10 s interval and a 10-fold proton gradient (pHi=6.5 and pHo=7.5) with addition of 2 mM MgCl2 (Methods). Values are mean±SEM, n=3 cells for each condition. Some error bars are smaller than symbols.
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- a, Representative proton current traces for hHv1 channels before (left), and in the presence of 1 mM EGTA (right), with steps of 20 mV increments from −80 mV to +40 mV.
- b, G-V for hHv1 in the absence (U) or presence of 1 mM EGTA (0). hHv1 channels showed a 40±3 mV shift after exposure to 1 mM EGTA from 15±1 mV to −25±2 mV. Curves are fitting to the Boltzmann equation.
- c, 500 μM Alb (red trace) was applied after control pulses after pre-activation with 1 mM EGTA (black trace) and shows attenuated activation of the proton current.
hHv1-R205N and hHv1-R211S channels, carrying changes in the first and the third voltage sensing Arg residue in S4, respectively, were expressed in HEK293T cells, and studied using whole-cell voltage clamp with 1.5 s pulses to 0 mV (hHv1-R205N) or +40 mV (hHv1-R211S) from a holding voltage of −60 mV with 10 s interval with a 10-fold proton gradient (pHi=6.5 and pHo=7.5) and divalent cation-free bath solution. After control pulses (black trace), 75 μM Alb (red trace) was applied.
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- a, The R205 mutation fully eliminate activation by 75 μM Alb
- b, The mutation of R211 did not alter Alb-activation showing an approximately 5-fold increase in current (n=3).
This example demonstrates that methods and compositions as provided herein are effective for inhibiting, ameliorating or preventing PMN-mediated inflammation in vivo.
This research addresses a specific unmet medical need, the absence of an effective medical therapy for Acute Respiratory Distress Syndrome (ARDS), a disorder that is fatal in 40% of patients (and is seen, for example, in patients with SARS-CoV-2 infection). The work is broadly impactful because the white blood cells called neutrophils (PMN) that damage the lungs in ARDS also cause other acute and chronic inflammatory diseases. In the last period, we showed that the human voltage-gated proton channel (hHv1) in PMN is essential to produce the inflammatory response, and provided herein are compositions and methods to confront ARDS by a novel strategy: targeting hHv1 in PMN, both directly and by blocking a natural activator of hHv1 we describe here to be required for normal hHv1 operation, thereby offering two ways to confront the disease at the earliest stage before it becomes a more complex disorder.
Treatments to prevent or ameliorate ARDS have resisted efforts for decades, in-part because many insults can lead to the disease, and once underway the pathology becomes complex, dysregulating a wide array of downstream processes8. Our demonstration that hHv1 in human PMN is required to initiate and sustain the release of inflammatory mediators5, and the essential role of endogenous Alb we describe here, compels us to investigate inhibition of hHv1 as a therapy for ARDS either by direct block of hHv1 and/or suppression of the newly-discovered, Alb-activation of hHv1.
PMN are the most abundant phagocytes in humans, accounting for 50-60% of the circulating white blood cells, and they are usually the first cells to arrive at sites of infection. Perhaps, the most dramatic example of damage caused by the PMN inflammatory response is ARDS, a disease that is fatal in approximately 40% of cases8. PMN are recognized as a hallmark feature and the driver of ARDS, see
C6 is a de novo inhibitor isolated from over 106 combinatorial variants of voltage-sensor directed neurotoxins, and C6 allowed us to demonstrate that hHv1 is required for the normal operation of human sperm and the PMN innate immune response. This was not feasible when the available hHv1 inhibitors were promiscuous and of low affinity (Zn2+ ions29, HaTx130,31, and guanidinium derivatives32). As the studies show, Alb is an endogenous hHv1 activator that is required for human sperm and PMN to operate. Further, we present a testable structural-mechanistic model for hHv1 activation by Alb whereby the two S3-S4 hHv1 sensor loops bind to sites in the Alb domain II. This allowed us to rationally-design a new agent (L*), a peptide that suppresses Alb-activation and ROS production by human PMN.
We reveal as well that oxidized metabolites of Alb formed naturally at sites of inflammation by PMN are even more potent activators of hHv1 and ROS release. Provided is in vivo support for our underlying hypothesis that suppression of hHv1 can be therapeutic: C6 administration inhibits pulmonary compromise and inflammatory mediator release in an acute lung injury mouse model.
Alb was implicated in the deleterious systemic inflammatory responses mediated by PMN after cardiopulmonary bypass and in periodontal disease44,45 When 75 μM Alb is added to the bath, hHv1 currents in HEK293T cells increase approximately 5-fold at 0 mV due to an approximately 8-fold acceleration of activation, approximately 3-fold slowing of deactivation, and a shift in the V1/2 of approximately −23 mV (
Important loop residues (
To model the Alb-hHv1 complex in the absence of high resolution information, in silico docking was performed (HPEPDOCK)51 using the crystal structure of Alb (PDB 1BMO)46 and the 11-residue binding epitope we identified in S3-S4 loop of hHv1 (F190 to L200). Initial docking predicted two sites in Alb, separated by approximately 27 Å, as putative hot spots for interaction. This distance is comparable to that between H193 residues in the two hHv1 subunits (approximately 26 Å) in the 3D model based on electron paramagnetic resonance (EPR) spectroscopy1. Consistent with the mutation that eliminated Alb activation (
A structural model and mechanistic hypothesis for Alb activation (with E Perozo). A model of the Alb-hHv1 complex was generated based on the residues identified as critical for binding by voltage-clamp and FRET (
To measure ROS release we use a luminol-amplified chemiluminescence assay for superoxide anion (O2*−)74. When we apply an immune stimulus (1 μM fMLP75) without Alb, a transient rise and decay in ROS release is observed (
This example demonstrates that methods and compositions as provided herein are effective for inhibiting, ameliorating or preventing PMN-mediated inflammation in vivo.
Here, we demonstrate that a mouse model for bacterial pneumonia (LPS administration into the lungs) and sepsis is suppressed by C6 peptide. The pathologic stimulus is LPS delivered into the lungs and the treatment is C6 given intravenously.
MethodC57BL/6 mice aged 9-12 weeks (half male half female) were used for experiments. Lipopolysaccharide (LPS, 10 mg/mL, 50 μL) was administrated intratracheally (IT) to induce acute lung injury. 5 hours post LPS induction, 2.5 mg/kg C6 peptide was administrated intravenously (IV) for treatment. 24 post LPS induction, another dose of C6 peptide (2.5 mg/kg) was administrated IV in order to supplement the bioavailability of C6. DMSO was used as negative control for treatment. 48 hours post LPS induction, the lung compliance was determined using the Flexivent system, then mice were sacrificed and other lung injury parameters were quantified. Briefly, alveolar-capillary barrier dysfunction was accessed based on the total bronchoalveolar lavage (BAL) fluid protein concentration and total BAL cells counts, inflammation level was assessed by quantification of BAL inflammatory cytokines levels, and ROS production level in BAL fluids.
Results C6 Reduces the Body Weight Loss of Mice Induced by LPS (FIG. 1).ARDS is marked by PMN-mediated inflammation and acute respiratory failure, features that are reflected in the LPS-induced mice including recruitment and activation of PMNs to the lungs, damage to the alveolar permeability barriers, and a decrease in lung compliance. Administration of C6 improves the lung compliance of mice induced by LPS (
The healthy lung is structured to facilitate rapid gas transfer across the distal alveolar-capillary unit. The alveoli consist of a continuous monolayer of endothelial and epithelial cells that restrict the passage of solutes and fluid but allow diffusion of carbon dioxide and oxygen from the vasculature. Large numbers of PMNs traverse the alveolar capillaries. During ARDS, alveolar endothelial cells and resident macrophages secrete chemokines to recruit circulating PMNs to migrate into the alveoli damaging the alveolar-capillary barrier. Administration of C6 decreases the total cell accounts (including neutrophils) in BAL fluid (
Protein-rich fluid influx into the lung after the damage of the alveolar-capillary barrier in ARDS. Administration of C6 decreases the total protein concentration in BAL fluid (
When activated, PMNs release ROS, which plays a role in killing pathogens, but also injures the lung leading to accumulation of alveolar edema, poor gas exchange and arterial hypoxemia.
Administration of C6 decreases the total ROS production by cells in BAL fluid (
The levels of proinflammatory cytokines are elevated in BAL fluid in ARDS patients. C6 prevented LPS-induced accumulation of proinflammatory cytokines, including IL-6, IFN-7 and IP-10 in the BAL fluid (
Before the development of C6, known hHv1 inhibitors were pharmacologically promiscuous or of low affinity (8-10). Synthesized C6 inhibits hHv1 by binding with positive cooperatively to the external channel residues linking the S3 and S4 transmembrane spans (S3-S4 loop), one peptide on each loop, and holds the voltage sensor in a conformation that favors channel closure (
Here, we demonstrate that as with natural gating modifier toxins, C6 dissociation is accelerated by depolarization (11, 12). As expected, mutations in the S4 of hHv1 that favor the open channel state show a lower affinity for C6, while mutants that favor channel closure enhance C6 inhibition. Using point mutations, we identity five residues in the hHv1 S3-S4 loop that alter the free energy of blockade (ΔΔG) by more than 2 kcal/mol and that we therefore characterize as important for C6 binding. Taking advantage of a membrane-tethered toxin method (13), we scanned 35 non-cysteine residues in tethered C6 (T-C6) and identified seven that also significantly decrease affinity. We also show that C6 partitions most readily into lipid membranes that contain negatively charged phospholipids. Based on these screening results, molecular dynamics (MD) simulations were carried out to predict energetically important residue-residue interactions in the binding of C6 to hHv1 and the potential role of the lipid bilayer in inhibition. Three predicted C6-hHv1 interaction pairs were confirmed by thermodynamic mutant cycle analysis, supporting the presented structural model of the complex. This model undepins a gating modification mechanism through which binding of C6 holds the S4 helix of hHv1 in the closed “down” conformation. hHv1 operates differently than well-studied voltage-gated channels like those for K+, Na+ and Ca2+ ions that have one central pore and four peripheral voltage sensors. Our findings assess the operation and ICK toxin inhibition of H+ channels that are dimeric, with two pores, one within each voltage sensor.
Natural bivalent toxin peptides, such as DkTx and Hi1a, isolated from spider venoms have been shown to modify the function of TRPV1 and ASIC1a channels, respectively. The natural toxins act with superior affinity due to their extremely slow dissociation rate (14, 15) and are comprises of two non-identical ICK motifs that target two adjacent binding epitopes in the channel (16). Guided by the C6-hHv1 complex model, we constructed a homobivalent C6 (C62) linking two C6 peptides via a 10-residue linker and show that it fully inhibits hHv1 at +40 mV with a Ki of 630 μM. The importance of blocking the channel at positive potentials is clear when considering neutrophils that depolarize during the inflammatory respiratory burst to +58 mV (17), a voltage where monomeric C6 has low affinity. C62 offers a powerful tool for basic studies of hHv1 and serves with the state dependent blocker C6 as a lead for studying treatment of diseases where the channel contributes to pathology, including pulmonary damage by leukocytes in pneumonia and acute respiratory distress syndrome (an inflammatory lung disease that is lethal in 40% of patients) (18), ischemic stroke (19), cancer (20), and both neuropathic and inflammatory pain (21, 22).
Materials and Methods Molecular BiologyHuman Hv1 (NM_001040107) tagged with a teal fluorescent protein (TFP) was constructed using gBlock gene fragments and inserted into pMAX+ vector, as reported (1). Per our prior reports (13), T-C6 was constructed by replacing the sequence of lynx1, a natural tethered nicotinic acetylcholine receptor peptide modulator, by the sequence of C6 (with N-terminus at front) in frame between the trypsin secretory signal sequence and a six residues flexible linker containing Gly-Asn repeat. The whole sequence of T-C6 was cloned into the pCS2+ plasmid vector that has an SP6 promoter for in vitro transcription of cRNA. T-C646 has a 46 residues flexible linker in which a c-Myc epitope tag is incorporated. T-C62 was constructed in the backbone of T-C6 by replacing the C6 sequence with nucleotides encoding two C6 peptides linked by a flexible linker of either 5 or 20 Gly-Asn repeats using gBlocks. Point mutations of hHv1 and T-C6 were introduced using the QuikChange Site-Directed Mutagenesis Kit. The sequences of all constructs were confirmed by DNA sequencing.
Toxin Peptide Synthesis and Purification: C6 peptide (GenBank: AZI15804) and its variants, including C6-extN and C6-extC, were synthesized by CSBio. Peptides were dissolved in external solutions for electrophysical recordings before use.
Whole-cell patch-clamp: Proton currents passed by hHv1 were recorded in whole cell mode using an Axopatch 200B amplifier. Stimulation and data collection were done with a Digidatal322A and pCLAMP 10 software (Molecular Devices). Cells were perfused with an external solution comprised of 100 mM HEPES, 100 mM NaCl, 10 mM glucose at pH 7.5. Pipettes with resistances between 3-5 MΩ were filled with 100 mM Bis-Tris buffer, 100 mM NaCl, and 10 mM glucose at pH 6.5. Capacitance was subtracted online. Sampling frequency was 10 kHz with filtering at 1 kHz. C6 block and unblock, current-voltage relationships, conductance-voltage relationships and the dose response curves were determined as described in the SI Materials and Methods.
Two Electrode Voltage Clamp: Xenopus laevis stage VI oocytes were selected and injected with 10 ng of cRNA encoding hHv1 in the laboratory pMAX+ vector, as before (13, 46). To study the blocking effect of T-C6 (or variants), cRNAs for T-C6 (or variants) and hHv1 were mixed and co-injected into the oocytes. Recording solution was 60 mM NaCl, 1 mM MgCl2, 2 mM CaCl2), 120 mM HEPES, 40 mM sucrose at pH 7.2. To prevent changes in intracellular pH due to the proton efflux, oocytes were injected with 50 nL 1 M HEPES (pH=7.2), to produce approximately 100 mM HEPES in the cytosol, 30 min before recording. Currents were recorded 2 days after cRNA injection using an Oocyte clamp amplifier OC-725C (Warner Instruments) with electrodes filled with 3 M KCl and resistances of 0.3-1 MΩ.
Enzyme-linked Immunosorbent Assay: Surface expression of T-toxins were quantitated using ELISA as previously described (13). Oocytes injected with cRNAs of T-C6 variants bearing the c-Myc tag were blocked with BSA and then bound with c-Myc-Tag monoclonal antibody HRP (1 μg/mL) (Invitrogen). Oocytes were washed and then incubated with 50 μL of 1-Step Ultra TMB-ELISA solution (Thermo Fisher Scientific). The reaction was stopped by adding 50 μL of 2 M H2SO4. Surface ELISA signals were quantitated at 450 nm.
Toxin Depletion Assay: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) were dried from a chloroform solution under a nitrogen stream. The dried lipids were rehydrated in a buffer comprised of 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2), 1 mM EDTA at pH 7.5. The resulting dispersions were extruded through 100 nm pore size polycarbonate filters (Millipore) and mixed at POPC:POPG=3:1 ratio to form large unilamellar vesicles (LUVs). Varying concentrations of POPC:POPG LUVs or POPG only LUVs were added to an aqueous C6 solution (final concentration of C6 was 50 μM in 200 L) and incubated with gentle agitation for 30 min at room temperature. LUVs were separated by high-speed centrifugation (100,000 g, 20 min). C6 toxin remaining in the aqueous phase was determined using HPLC. Depletion experiments with native cell membranes are described in the SI Materials and Methods.
Simulation Systems and Molecular Dynamics Simulations: The homology model of C6 was constructed with the MODELLER program (47) using the NMR structure of the natural inhibitor cysteine knot (ICK) spider toxin Mu-agatoxin-Aa1a as the template (PDB: 1EIT) as described in the SI Materials and Methods. The bilayers of POPC:POPG (3:1) or pure POPC lipids were built using the web service CHARMM-GUI (48). The six cysteine residues of C6 were patched to form three disulfide bonds using the psfgen plugin of VMD (49). The C6 was placed on the extracellular side of the bilayer, and then solvated with 100 mM KCl solution using VMD. The two final C6-membrane systems were minimized and equilibrated using NAMD (50), and then simulated for 1 μs on the special purpose computer Anton2 (51). The starting system of the C6-hHv1 complex was constructed using the equilibrated C6 structure and the transmembrane region (residues: G90 to 1216) of the resting state dimeric hHv1 model adopted from our previous study (23) using VMD. hHv1 homology model was inserted into a bilayer of POPC:POPG (3:1) lipids, and two C6 molecules were placed on the extracellular side of hHv1 with the F28 containing loop (loop 4) facing the lipids and the potential binding interface facing the S3-S4 loop of each monomer of the channel. A stepwise target MD simulations protocol (13) was used to refine the complex with distance restraints between C6-lipids and C6-hHv1 using NAMD as described in the SI Materials and Methods. The “up” state of the hHv1 model was built by moving the S4 helix of the “down” state one helical turn outward, while leaving the S1-S3 helices unchanged as in our previous study (23). The C62 model was built using MODELLER as described in the SI Materials and Methods. The C62 model was inserted into the C6-hHv1 system to replace the two C6 monomer and then simulated as described in the SI Materials and Methods.
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- (A) Cartoon showing two C6 peptides binding on two subunits of a hHv1 channel.
- (B) Representative H+ current traces for hHv1 channels before (left), and in the presence of 250 nM C6 (right) with steps of 20 mV from −60 to +40 mV. The peak current at the end of the step was used to determine the extent of block.
- (C) Current-voltage relationships for hHv1 in the absence (▪) or presence of 250 nM C6 (●). C6 inhibition was greater at more hyperpolarized potentials with a maximal blockade of 90% at −20 mV, which decreased to 77% at 0 mV.
- (D) Dose-response relationships for C6 inhibition of hHv1 studied at +40 mV (0), +20 mV (●) and 0 mV (●). The inhibition constant Ki of C6 for hHv1 channels at +40 mV, +20 mV and 0 mV were estimated from the fit to Eq. 1 to be 30.9±3.4 nM (with a Hill coefficient h=0.48±0.04), 4.9±0.8 nM (h=0.69±0.05), and 1.5±0.2 nM (h=1.22±0.17), respectively (Table 1, Example 4). Some error bars are smaller than symbols.
- (E) The time course for block and unblock of hHv1 on acute application (red bar) and washout of 250 nM C6 recorded at +40 mV (●), +20 mV (●) and 0 mV (●). The association rate constants (kon) and dissociation rate constants (koff) were approximated from the kinetics of block and unblock using Eqs. 3 and 4, relationships derived for bimolecular binding reactions, because the kinetics were well-fitted with single exponentials (Table 1).
- (F) Effect of voltage on C6 blocking kinetics. The kon was insensitive to voltage, whereas koff and Ki were responsive. Blocking parameters, kon (▴), koff (♦), Ki (●) were normalized to its value at 0 mV. Ki, kon and koff were determined as described in panel D and E. Error bars are smaller than symbols.
- (G) Conductance-voltage relationships (G-V) for hHv1 (m), hHv1-R205N (A) and hHv1-R21 1S (V). Curves are fitting to the Boltzmann equation (Materials and Methods). The V1/2 of G-V of channels are reported in Table S1.
- (H) Dose-response relationship for C6 inhibition of hHv1-R205N studied at +40 mV. Insert: representative current traces with 1 μM C6 (red) or without C6 (black).
- (I) Dose-response relationship for C6 inhibition of hHv1-R211S studied at +40 mV. Insert: representative current traces with 1 μM C6 (red) or without C6 (black).
hHv1 expressed in Xenopus oocytes was studied by TEVC to assess T-C6 inhibition at equilibrium from a holding voltage of −60 mV with 1.5-s test pulses and a 10-s interpulse intervals, with symmetric pHo and pHi=7.2 maintained by injection of 50 nL 1 M HEPES into oocytes (Materials and Methods). Values are mean±SEM; n=12 cells for each condition.
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- (A) T-C6 was constructed as chimeric fusion protein with an N-terminal secretory signal sequence (SP; gray), the C6 sequence (red), a hydrophilic flexible linker (6 residues), and a C-terminal GPI membrane anchor targeting sequence (orange) (
FIG. S2 ). The colors in the schematic mark T-C6 components. - (B) Representative current traces (steps from −60 to +80 mV) for hHv1 channels without (CTL) or with 20 ng T-C6 cRNA coinjection.
- (C) hHv1 currents at +80 mV (Peak) and −60 mV (Tail) with T-C6 (20 ng of cRNA) normalized to the unblocked condition (CTL).
- (D) T-C6 linker variants with hydrophilic flexible linkers range from 6 to 66 residues show different extent of inhibition to hHv1. The average proton currents at +80 mV with T-C6 linker variants (20 ng of cRNA) were normalized to the unblocked condition (CTL). Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment reverses blockade by T-C6 (6 residues linker).
- (E) Concentration-response for T-C6 inhibition of hHv1 currents at +80 mV normalized to the unblocked condition (CTL).
- (A) T-C6 was constructed as chimeric fusion protein with an N-terminal secretory signal sequence (SP; gray), the C6 sequence (red), a hydrophilic flexible linker (6 residues), and a C-terminal GPI membrane anchor targeting sequence (orange) (
hHv1 was expressed in oocytes and inhibition by T-C6 and variants were studied by TEVC after coinjection of 20 ng of the cRNAs. hHv1 currents at +80 mV with T-C6 variants were measured as in
A depletion assay (Materials and Methods) was used to determine the partitioning of C6 into lipid membranes. Large unilamellar vesicles (LUVs) were prepared with POPC:POPG (3:1) and incubated with C6 solution. C6 peptide remaining in the aqueous phase was quantitated by HPLC (
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- (A) Ratio of bound/free C6 (red) or AgTx2 (black) plotted as a function of LUVs concentration. Values are mean±SEM; n=3 for each condition. Some error bars are smaller than symbols.
- (B) Surface and ribbon representations of the homology model of C6. The heavy sidechain and Ca atoms of key residues derived from screening mutagenesis (
FIG. 4 ) are highlighted in stick and surface representation. Three disulfide bonds (C4-C18, C11-C23, and C17-C34) that demonstrate a classic ICK scaffold are highlighted in stick representation. Basic, polar and hydrophobic residues are colored blue, green and yellow, respectively. - (C) Time series of the z coordinate of the center-of-mass of Ca atoms of the six cysteines (orange line) of C6, phosphate atoms in the upper and inner lipid leaflets (gray lines), and the heavy sidechain atoms of F28 (red line) and W38 (blue line) of C6, respectively, in the molecular dynamics (MD) simulations system with a POPC:POPG lipid bilayer.
- (D) Snapshot of C6 partitioning into a POPC:POPG lipid bilayer using its hydrophobic motifs in the MD simulations. Phosphate atoms of lipids are shown in sphere representation (only part of the lipids are shown for clarity). The two hydrophobic residues of C6, F28 and W38 (yellow), inserting into the lipid bilayer are shown in stick and surface representation. Three disulfide bonds are shown in stick representation (cyan).
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- (A) The C6-hHv1 complex in two orientations showing two C6 molecules (orange) bind to the two subunits (cyan and blue) of an intact dimeric hHv1 channel. The transmembrane helices S1 and S4 form the dimer interface. The horizontal gray lines indicate the position of the membrane boundary.
- (B) Close-up view of the interaction interface between one C6 and one hHv1 subunit in the structural model. F28, M33 and W38 (yellow side chains) of C6 form hydrophobic interactions with L200, H193 and V187 (yellow side chains) of hHv1, respectively. K31 and R36 (blue side chains) of C6 form salt bridges with E196 and E192 (red side chains) of hHv1, respectively.
Peptides of C6, C6-F28A, C6-K31A, and C6-R36A were synthesized and tested on hHv1, hHv1-V187A, hHv1-E192A, hHv1-Q194A, hHv1-E196A and hHv1-L200A in HEK293T cells using whole-cell patch clamp (at +40 mV) as described in
-
- (A-F) Dose-response curves for the inhibition of WT and mutant channels by C6 and variants. Ki were determined by fitting the curves to the Hill equation (Eq. 1). Ki of all combinations are reported in Table S3.
- (G) A thermodynamic cycle for mutations C6-R36A and hHv1-E192A, showing large coupling energy. Each corner of the cycle represents the Ki of an indicated toxin-channel combination pair. Q is defined in Eq. 8, and the coupling energy is calculated using Eq. 9; these are approximations using relationships derived for bimolecular binding reaction.
- (H) In contrast to panel G, a cycle for C6-K31A with hHv1-E192A shows negligible coupling energy (lnΩ≈0), despite the large energetic effect of the C6-K31A mutation alone (
FIG. 4 ). - (I) Coupling energy values for the indicated combinations of peptide C6 variants and hHv1 mutations. Three toxin-channel pairs, C6-F28 and hHv1-L200 (ΔΔG=1.5 kcal/mol), C6-K31 and hHv1-E196 (1.6 kcal/mol), C6-R36 and hHv1-E192 (2.7 kcal/mol), show large coupling energies.
Inhibition by T-C62 of hHv1 was studied in oocytes by TEVC, as in
-
- (A) T-C62 was constructed using T-C6 and replacing the encoding sequence for C6 with two C6 connected by flexible linkers with either 10 or 40 residues (Materials and Methods).
- (B) Inhibition of hHv1 currents in oocytes by T-C6 and T-C62 linker variants (20 ng cRNA) studied at +80 mV and shown normalized to the unblocked condition (CTL).
- (C) Conjugation of C6 peptides using Sortase A transpeptidase enzyme. C6 peptides with C-terminal (C6-extC) or N-terminal (C6-extN) extensions were synthesized. Sortase A cleaves between Thr and Gly in the recognition sequence at the C-terminus of C6-extC and catalyzes the formation of an amide bond with the nucleophilic polyglycine at the N-terminus of C6-extN.
- (D) Representative H+ current traces for hHv1 channels in HEK293T cells before (Left), and in the presence of 10 nM C62 peptide (Right) with steps of 20 mV from −60 to +40 mV. The peak current measured at the end of depolarization was used to determine the extent of block.
- (E) The time course for block and unblock of hHv1 in HEK293T cells on acute application (red bar) and washout of 10 nM C62 peptide recorded at +40 mV.
- (F) Dose-response relationships for C62 peptide inhibition of hHv1 in HEK293T cells at +40 mV. The inhibition constant Ki of C62 for hHv1 channels was estimated from the fit to Eq. 1 to be 631±52 μM with a Hill coefficient of 1.01±0.03.
- (G) The homology model of C62 and the ‘down’ state dimeric hHv1 model were used to build a structural model of C62-hHv1 complex using MD simulations (Materials and Methods). The C62-hHv1 complex in two orientations shows two C6 epitopes (orange) connected by a 10-residue peptide linker (LPATGGGGGG) (SEQ ID NO:14) binding simultaneously to the two hHv1 subunits (cyan and blue). The horizontal gray lines indicate the position of the membrane boundary.
State Dependent Inhibition of hHv1 by C6
We previously reported the construction of an ICK scaffold phage-display library with over 1 million variants (1). The ICK scaffold is widely seen in venom toxins that bind to the VSDs of a variety of voltage-gated channels and modify the movement of the voltage sensors. Toxin variants possessing the ICK scaffold are stabilized by six cysteines that form three intramolecular disulfide bonds and are expressed on the phage surface via encoding into coat protein pIII allowing for screening. C6 was isolated by sorting the library on purified hHv1 channels. Stably bound phage were enriched by washing to remove weak and non-specific interactions, followed by elution, amplification and rebinding through five rounds of panning. The most enriched phagemids expressed C6 and were increased approximately 65,000-fold from starting abundance. Subsequent characterization using smTIRF microscopy showed that the C6 peptide binds to hHv1 expressed in HEK293T cells with a Kd of 0.75 nM at −49 mV (a potential favoring the channel closed state), whereas the Ki is attenuated to 31 nM at +40 mV as determined using whole-cell patch clamp to study block of current passed by the open channels (1). Arguing for positive cooperativity of C6 binding at resting membrane potential, such that binding of the first C6 on the dimeric channel facilitates binding of a second C6 on the other subunit, we found the dose-binding curve was well-fitted using the Hill equation with a coefficient of 1.5 and, further, a fit using the Monod-Wyman-Changeux relationship estimated the first C6 to bind with approximately 12-fold lower affinity than the second C6 at the two allosterically coupled sites (1).
To explore the mechanism of voltage-dependent inhibition of C6, hHv1 channels were expressed in HEK293T cells and studied by whole-cell patch clamp with a 10-fold proton gradient (pHi=6.5 and pHo=7.5), as before (1). Here, by assessing C6 blocking parameters, we show that C6 affinity for hHv1 decreases on membrane depolarization largely due to acceleration in the C6 dissociation rate. This is recapitulated by point mutations of “gating charge” residues in the hHv1 S4 helix that respond to voltage to mediate channel opening.
At +40 mV, 250 nM C6 decreased hHv1-mediated outward H+ currents by approximately 45% (
The dose-response for C6 block of hHv1 at +20 mV and 0 mV plotted similarly (
The Ki for toxin block can also be estimated by the ratio of the first-order dissociation rate constant (koff) and the second-order association rate constant (kon) according to Eq. 2.
Furthermore, kon and koff are related to the association constant (□on) and dissociation constant (Doff) derived from single exponential fits of the time courses for toxin block and unblock by Eqs. 3 and 4.
Thus, the time course of wash-in and washout of 250 nM C6 can be fitted with single exponentials, allowing approximation of the time course of inhibition, kon=3×105±0.8×105 M−1s−1, and recovery, koff=0.022±0.001 s−1 at +40 mV (
Comparing the kinetics of C6 wash off at various potentials made it readily apparent that toxin dissociation was strongly dependent on voltage (
The S4 helix of hHv1 contains three conserved Arg residues (R205, R209, R211) which confer the channel with sensitivity to voltage (4). These conserved Arg form salt-bridges with acidic residues in the S1-S3 helices during state transitions similar to conventional voltage-gated channels and enzymes (23-26). We neutralized two Arg residues in the hHv1 S4 and observed conductance-voltage (G-V) relationship shifts consistent with the hypothesis that R205 helps maintain a closed state of hHv1, while R211 contributes to stabilizing an open state (2, 27). Thus, the hHv1-R205N mutation shifted V1/2 by approximately −57 mV, favoring channel opening, while the hHv1-R211S mutation shifted the V1/2 by approximately +33 mV, favoring the closed state (
Mapping the residues in the hHv1 S3-S4 loop critical for C6 binding: We showed that C6 binds to the external S3-S4 loop of hHv1 by “transplanting” the hHv1 loop region (I183 to L204) into the C6-insensitive channel CiHv1, conferring the chimeric channel with toxin sensitivity similar to that of WT hHv1 (1). To explore the role of individual S3-S4 loop residues in C6 binding, 22 loop residues (I183 to L204) were individually mutated and C6 blockade of the mutant hHv1 channels were studied at +40 mV using whole-cell patch clamp (
By consensus, a value of 2 kcal/mol is used as the cutoff for ΔΔG to identify residues most likely to be engaged in statistically relevant energetic interactions at protein-protein interaction interfaces (28, 29). Thus, the observed large free energy changes in C6 binding suggest that these five channel residues may mediate direct interaction with C6. We also found three mutations (hHv1-D185C, hHv1-G199C and hHv1-L203C) that enhanced C6 inhibition more than 4-fold (
Identification of critical toxin residues using T-C6 variants: The in vitro synthesis and folding of venom toxin peptides can be technically challenging, costly, and time consuming. To facilitate characterization of C6 by mutational screening, we employed a strategy whereby C6, or its variants, were expressed from a gene in tethered form on the extracellular surface of cells via a glycosyl phosphatidyl inositol (GPI)-anchored membrane-embedded leash, as we have before with other toxins (13). The construct included a signal peptide sequence, C6 or its variants, and a flexible linker followed by a GPI targeting sequence (
C6 has 41 residues with three disulfide bonds that maintain the ICK toxin scaffold. To identify the C6 residues that might mediate direct interaction with the S3-S4 loop of hHv1 (or membrane lipids around the channels), we generated T-C6 point mutations at the 35 positions that were not Cys (
To confirm that the effects of mutations were not due to changes in surface expression, we constructed the same mutations in T-C6 with 46-residue linker (T-C646), which carries a c-Myc epitope, allowing comparison of the surface levels of T-C646 and variants by ELISA, as we have described (13). Injection of 20 ng T-C646 cRNA inhibited 39% of hHv1 outward current (
C6 partitioning into cell membranes: ICK toxins like HaTx and ProTx2 from spiders inhibit voltage-gated channels by first partitioning into the plasma membrane followed by direct interaction with VSDs (31, 32). We studied the partitioning of C6 into large unilamellar vesicles (LUVs) composed of a 3:1 mixture of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) phospholipids using a toxin depletion assay (33). Varying amounts of LUVs were incubated with C6 in solution, and liposome-bound C6 was separated by ultracentrifugation. C6 remaining in the aqueous phase was quantified using reverse-phase, high-performance liquid chromatography (HPLC). Roughly 50% of C6 peptide partitioned into 0.5 mM LUVs (
Kp=[Fp/(1−Fp)][W]/[L] Eq. 7
The Kp of C6 is comparable to that reported for HaTx (5.3×105) (31), suggesting C6, like other natural ICK toxins, interacts with membrane lipids (31, 32). To confirm that C6 partitions into native membranes, we examined the ability of intact Xenopus oocytes and HEK293T cells to deplete C6 from physiological solutions (Materials and Methods). When cells are agitated gently in a solution containing C6, we observed depletion of 34% and 67% of C6 from the aqueous phase by oocytes and HEK293T cells, respectively. In contrast, others have shown that less than 1% of AgTx2 toxin peptide was depleted by the same number of oocytes (31).
A homology model of C6 and its interaction with lipids: Mu-agatoxin-Aa1a is a spider ICK toxin that activates insect voltage-gated Na+ channels (35) and shows 40% identity to the C6 peptide sequence. The NMR structure of Mu-agatoxin-Aa1a (PDB: 1EIT) was chosen as a template to generate a C6 homology model (Materials and Methods). Our C6 model employs a standard ICK scaffold with four solvent accessible loops protruding from a disulfide-bonded globular core (
One face of C6 stands out as containing six out of the seven critical residues (K24, F28, K31, M33, R36, and W38) where mutations to Ala increased Ki values >43-fold (ΔΔG>2 kcal/mol,
To investigate the interaction of C6 and lipids, we constructed an all-atom system of C6 and a fully hydrated lipid bilayer of POPC:POPG (3:1) as used in the toxin depletion assay (
A proposed model of C6 binding to the hHv1 closed state: Based on our knowledge of the orientation of C6 partitioning into lipids (
Validation of the model by point mutations: Two charge reversal experiments inspired by our MD simulations further support the model of the C6-hHv1 complex by meeting the expectations for salt-bridges between hHv1-E192 and C6-R36, and between hHv1-E196 and C6-K31. When the side chain hHv1-E192 was reversed in charge by mutation to Lys (producing hHv1-E192K), inhibition by T-C6 was reduced approximately 52-fold. When the predicted C6 charge partner, R36, was reversed to Glu (producing T-C6-R36E), toxin inhibition of hHv1-E192K was restored. As a control, two other point mutations, T-C6-K24E and T-C6-K31E were studied with hHv1-E192K channels and they did not restore inhibition. Similarly, T-C6 inhibition was reduced by approximately 12-fold with hHv1-E196K channels, block was restored by the counter mutation T-C6-K31E, but the control changes T-C6-K24E and T-C6-R36E did not restore inhibition. These ‘side chain swaps’ validate the model position of hHv1-E192 and hHv1-E196 near C6-R36 and C6-K31, respectively.
Pairwise interactions confirmed by mutation cycle analysis: We used thermodynamic mutant cycle analysis to verify pairwise interactions suggested by the C6-hHv1 complex model, an approach brought to the fore by mapping the binding site of scorpion toxins in the pore of the K+ channels (38). The interaction between residues on the channel and toxin are identified by making channel mutations (Mut) and toxin variants (Var) and measuring the Ki for different combinations of modified and unmodified pairs. The coupling energy ΔΔG between Mut and Var can be calculated from the measured Ki according to Eqs. 8 and 9. The size of the ΔΔG quantifies the strength of interaction between the Mut and Var.
Ω=(Ki WT C6, WT hHv1×Ki C6 variant, hHv1 mutation)/(Ki WT C6, hHv1 mutation×Ki C6 variant, WT hHv1) Eq. 8
ΔΔG=RT lnΩ Eq. 9
We synthesized peptides for three C6 variants, C6-F28, C6-K31A and C6-R36A for the mutant cycle analysis. On the channel, we investigated mutations of five residues, hHv1-V187A, hHv1-E192A, hHv1-Q194A, hHv1-E196A and hHv1-L200A in the S3-S4 loop (
A designed bivalent C6 inhibits hHv1 fully at depolarized voltages: In the C6-hHv1 model, the two VSDs are stabilized by two C6 peptides (
Bivalent toxin peptides are difficult to synthesize and fold in vitro due to their long and disulfide-rich sequences. Sortase A is a bacterial thiol transpeptidase that can ligate peptides with a C-terminal recognition sequence (LPXTG) (SEQ ID NO:15) to peptides containing an N-terminal polyglycine sequence. Because the reaction conditions do not require a reduced environment, and show outstanding efficiency, Sortase A has been used to link toxin peptides (40). Here, we synthesized two C6 peptides, C6-extC and C6-extN, with 6 additional residues (LPATGG (SEQ ID NO:16) or GGGGGG (SEQ ID NO:17)) at C-terminus or N-terminus, respectively, and ligated the two peptides using Sortase A to create a bivalent C6 peptide with a 10-residue connecting linker, LPATGGGGGG (SEQ ID NO:18) (
A homology model of C62 and the ‘down’ state hHv1 was used to build a structural model of C62-hHv1 complex using MD simulations (Materials and Methods). The C62-hHv1 complex in two orientations showing two C6 molecules (orange) are connected by the linker of 10 residues and therefore both hHv1 subunits (cyan and blue) can bind a C6 epitope simultaneously (
The role hHv1 plays in acid extrusion and membrane potential compensation place the channel in the midst of important human physiological processes and classify it as a promising pharmacological target (3). Here, we study inhibition of hHv1 by the specific designer toxin C6, which we created to delineate the roles of the hHv1 in sperm and neutrophils, in order to better understand the structural-mechanistic basis for C6 action and thereby its potential as a drug lead. Because C6 targets the hHv1 VSDs, binding is voltage-dependent, and inhibition is only partial at both 0 mV and +40 mV with affinities of Ki=1.5 nM and 31 nM, respectively (
As observed for natural gating modifier toxins (11, 12), we demonstrate that depolarization induces a change from the closed channel conformation that binds C6 with high affinity to states that accelerate C6 dissociation, approximately 15-fold faster at test pulses to +40 mV compared to 0 mV (Table 1). In-keeping with this observation, a mutation in the S4 that favors the closed state (hHv1-R211S) slows C6 dissociation approximately 28-fold compared to WT channels, whereas a mutation that favors the open state (hHv1-R21 1S) accelerates C6 dissociation approximately 9-fold. Whereas C6 shows high affinity for the closed channel and positive cooperativity in binding, as the voltage becomes more depolarized, toxin association is slower, dissociation is faster, and negative cooperativity is observed, with a concomitant decrease in the extent of maximal blockade; thus, 250 nM C6 inhibits 90% of the current at −20 mV and 77% at 0 mV, due a decrease in the on-rate of 1.2-fold and an increase in the off-rate of 2-fold, across this small change in potential (
The mechanism of C6 blockade appears to have at least one similarity and two differences with natural ICK toxins that act on voltage-gated K+ and Na+ channels and TRPV1 (16, 32, 41). Like ICK spider toxins VSTx1 and HaTx (31, 37), C6 partitions into membranes by interaction with anionic lipids. Our MD simulations predict that basic residues in C6 form hydrogen bonds with lipid phosphate, ester oxygen atoms, and hydroxyl oxygen atoms in the head groups of anionic lipids. The electrostatic interactions of C6 basic residues and the anionic lipids stabilize adhesion of C6 onto the surface of the membrane, enabling formation of extensive hydrogen bond interactions of C6 residues and lipid polar head groups, and insertion of bulky C6 hydrophobic side chains into the hydrophobic core of the membrane lipids (FIG. 53D). These interactions restrain the orientation of C6 in the membrane and its binding interface with hHv1 channel.
The first apparent difference in the binding mechanism of C6 and natural ICK toxins is seen in the loop regions the toxins use for membrane partitioning. Here we compare C6 with findings for DkTx and ProTx2 visualized in complexes with TRPV1 and Nav1.7 channels, respectively, in a membrane-like environment using cryoelectron microscopy (16, 32). Whereas C6 uses two hydrophobic motifs locating in loop 4 (W25-F28) and C-terminus (V37-W38) for membrane partition (
A second difference is that C6 binds to residues in the S3-S4 loop of hHv1 that project out toward the lipid membrane (
Three pairs of toxin-channel interaction suggested by the model were validated by mutant cycle analysis and these serve to rationalize the voltage-dependent blocking mechanism. In the model, the S4 helices of hHv1 are in the “down” position, placing V187 and L200 adjacent to W38 and F28 in C6, respectively. The distance between E192 and E196 in S3-S4 loop (16 Å) matches the distance between K31 and R36 in C6 (15 Å), enabling a favorable electrostatic interaction (
Bivalent C6 is an advanced blocker for suppressing hHv1: We designed C6 to block hHv1 because previously reported blockers, including Zn2+ ions (42), HaTx (a spider toxin that blocks numerous K+ and Ca2+ channels) (9), and guanidinium derivatives that block from the inside of the membrane (10) are promiscuous and of low affinity, limiting their potential use in delineating the roles of hHv1 in physiology or as therapeutics. The case for hHv1 as a drug target is strong. While hHv1 is required to sustain the ROS production by neutrophils that is essential for clearance of bacterial infections by innate immune cells, excessive ROS production induces tissue damage, thrombosis and red blood cell dysfunction, contributing, for example, to the severity of COVID-19 (43). Further, in the central nervous system, ROS production by microglia was lower in an Hv1 knockout mouse than in wildtype, and this decreased the damage of ischemic stroke (19), and in a spinal cord injury mouse model the knockout prevented secondary damage after spinal cord injury (44). In addition, Hv1 is upregulated in some B-cell malignancies and breast cancers, and this is correlated with an increased metastatic potential (20, 45).
The ability of C6 to suppress sperm capacitation and sustained release of inflammatory mediators, including ROS, from neutrophils (1), is likely due to high affinity toxin binding to closed channels, the state favored at the resting membrane potential of the cells (Kd of 0.75 nM at −49 mV). While C6 stabilization of the closed state, and partial open channel blockade, might be sufficient for some therapeutic applications, we sought a more potent bivalent blocker that would bind to both hHv1 subunits simultaneously and used our C6-hHv1 model as a guide to design C62 (
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A number of embodiments of the invention have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Claims
1: A synthetic or recombinant peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic or recombinant peptide comprises:
- (a) an amino acid sequence QXHQFEXXXX (SEQ ID NO:1);
- (b) an amino acid sequence QAHQFEAXXX (SEQ ID NO:2);
- (c) an amino acid sequence QAHQFEALLL (SEQ ID NO:3), or
- (d) a homodimer or a heterodimer comprising a peptide of (a), (b), or (c),
- wherein X is any hydrophobic amino acid, and optionally the hydrophobic amino acid is glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) or phenylalanine (F).
2: A recombinant nucleic acid encoding a synthetic or recombinant peptide of claim 1.
3: An expression vehicle comprising or having contained therein a recombinant nucleic acid of claim 2, wherein optionally the expression vehicle comprises or is an expression cassette, a plasmid, an expression vector or a recombinant virus.
4: A cell comprising or having contained therein a synthetic or recombinant peptide of claim 1.
5: A pharmaceutical composition comprising: a synthetic or recombinant peptide of claim 1.
6: The pharmaceutical composition of claim 4, formulated as a liposome, a dendrimer or a nanoparticle; or formulated as an aerosol, a lyophilate or a powder.
7: A method for: (SEQ ID NO: 4) SSTCIPSGQPCADSDDCCETFHCKWVFFTSKFMCRRVWGKD (also called “C6”), or (SEQ ID NO: 5) GCKWYLGDCADSDDCCETFHCKWVFFTSKFMCRRVWGKD (also called “C5”),
- inhibiting intracellular alkalization, initiating capacitation, acrosomal reaction, and oocyte fertilization capabilities of human sperm,
- inhibiting the ability of neutrophils, or polymorphonuclear leukocytes (PMNs), to release reactive oxygen species (ROS),
- inhibiting or ameliorating neutrophil (PMN) contribution to an inflammatory response, or
- treating, ameliorating or preventing a neutrophil (PMN)-meditated inflammatory-related pathology,
- comprising:
- administering to an individual in need thereof:
- (a) a synthetic or recombinant peptide as set forth in claim 1, a recombinant nucleic acid encoding the recombinant peptide, an expression vehicle having contained therein the nucleic acid, a cell having contained therein the nucleic acid, or a pharmaceutical composition having contained therein or comprising the nucleic acid or synthetic or recombinant peptide, or
- (b) a synthetic peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide comprises a structure: (i) of about 10 to about 100 amino acids in length; (ii) having an amino acid sequence that comprises one or more toxin sequence elements, each of which has a length within a range of about 5 to about 18 amino acids in length; (iii) a peptide component has 0, 1, 2, 3, 4, 5, or 6 conserved cysteine residues of an ICK structural motif; (iv) has 0, 1, 2, or 3 disulfide bridges; and (v) has 0, 1, 2, or 3 beta strands,
- wherein optionally the synthetic peptide has a sequence comprising or consisting of:
- a homodimer comprising two peptides (SEQ ID NO:4) (also called “C6”) or two peptides (SEQ ID NO:5) (also called “C5”), or a heteroduplex of (SEQ ID NO:4) and (SEQ ID NO:5),
- or a sequence as described in U.S. patent application publication no: US 2019 0330285 A1.
8: The method of claim 7, wherein the neutrophil (PMN)-meditated inflammatory-related pathology is acute respiratory distress syndrome (ARDS),
- and optionally ARDS caused by a viral, bacterial or fungal infection; a sepsis; pancreatitis; trauma or injury; pneumonia; or, aspiration into the lungs.
9: The method of claim 7, wherein the synthetic or recombinant peptide, or the recombinant nucleic acid encoding the recombinant peptide, or the cell, or the pharmaceutical composition, is formulated and/or administered as a liposome, a dendrimer or a nanoparticle; or formulated or administered as an aerosol, a lyophilate or a powder.
10: A kit comprising a synthetic or a recombinant peptide of claim 1, a recombinant nucleic acid encoding the recombinant peptide, an expression vehicle having contained therein the nucleic acid, a cell having contained therein the nucleic acid, or a pharmaceutical composition having contained therein or comprising the nucleic acid or synthetic or recombinant peptide.
11. (canceled)
12: A synthetic or a recombinant peptide for inhibiting activity of a voltage-gated proton channel polypeptide (Hv1), wherein the synthetic peptide consists of:
- (a) an amino acid sequence QXHQFEXXXX (SEQ ID NO:1);
- (b) an amino acid sequence QAHQFEAXXX (SEQ ID NO:2);
- (c) an amino acid sequence QAHQFEALLL (SEQ ID NO:3), or
- (d) a homodimer comprising a peptide of (a), (b), or (c),
- wherein X is any hydrophobic amino acid, and optionally the hydrophobic amino acid is glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I) or phenylalanine (F).
13: The method of claim 7, wherein the neutrophil (PMN)-meditated inflammatory-related pathology is caused by a viral infection, and optionally the viral infection is an influenza or a coronavirus infection, and optionally the coronavirus infection is a COVID-19 infection.
14: The method of claim 7, wherein the synthetic or recombinant peptide is formulated and/or administered as a liposome, a dendrimer or a nanoparticle; or formulated or administered as an aerosol, a lyophilate or a powder.
15. (canceled)
Type: Application
Filed: May 27, 2022
Publication Date: Aug 1, 2024
Inventors: Steven A. GOLDSTEIN (Irvine, CA), Ruiming Zhao (Irvine, CA)
Application Number: 18/564,822
